Metabolic rewiring promotes anti-inflammatory effects of glucocorticoids.

Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood1. Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs.

Yield reduction caused by elevated temperatures and high nitrogen fertilization is mitigated by SP6A overexpression in potato (Solanum tuberosum L.).

Potatoes (Solanum tuberosum L.) are a fundamental staple for millions of people worldwide. They provide essential amino acids, vitamins, and starch - a vital component of the human diet, providing energy and serving as a source of fiber. Unfortunately, global warming is posing a severe threat to this crop, leading to significant yield losses, and thereby endangering global food security. Industrial agriculture traditionally relies on excessive nitrogen (N) fertilization to boost yields. However, it remains uncertain whether this is effective in combating heat-related yield losses of potato. Therefore, our study aimed to investigate the combinatory effects of heat stress and N fertilization on potato tuber formation. We demonstrate that N levels and heat significantly impact tuber development. The combination of high N and heat delays tuberization, while N deficiency initiates early tuberization, likely through starvation-induced signals, independent of SELF-PRUNING 6A (SP6A), a critical regulator of tuberization. We also found that high N levels in combination with heat reduce tuber yield rather than improve it. However, our study revealed that SP6A overexpression can promote tuberization under these inhibiting conditions. By utilizing the excess of N for accumulating tuber biomass, SP6A overexpressing plants exhibit a shift in biomass distribution towards the tubers. This results in an increased yield compared to wild-type plants. Our results highlight the role of SP6A overexpression as a viable strategy for ensuring stable potato yields in the face of global warming. As such, our findings provide insights into the complex factors impacting potato crop productivity.

Carbon usage in yellow-fleshed Manihot esculenta storage roots shifts from starch biosynthesis to cell wall and raffinose biosynthesis via the myo-inositol pathway.

Cassava is a crucial staple crop for smallholder farmers in tropical Asia and Sub-Saharan Africa. Although high yield remains the top priority for farmers, the significance of nutritional values has increased in cassava breeding programs. A notable negative correlation between provitamin A and starch accumulation poses a significant challenge for breeding efforts. The negative correlation between starch and carotenoid levels in conventional and genetically modified cassava plants implies the absence of a direct genomic connection between the two traits. The competition among various carbon pathways seems to account for this relationship. In this study, we conducted a thorough analysis of 49 African cassava genotypes with varying levels of starch and provitamin A. Our goal was to identify factors contributing to differential starch accumulation. Considering carotenoid levels as a confounding factor in starch production, we found that yellow- and white-fleshed storage roots did not differ significantly in most measured components of starch or de novo fatty acid biosynthesis. However, genes and metabolites associated with myo-inositol synthesis and cell wall polymer production were substantially enriched in high provitamin A genotypes. These results indicate that yellow-fleshed cultivars, in comparison to their white-fleshed counterparts, direct more carbon toward the synthesis of raffinose and cell wall components. This finding is underlined by a significant rise in cell wall components measured within the 20 most contrasting genotypes for carotenoid levels. Our findings enhance the comprehension of the biosynthesis of starch and carotenoids in the storage roots of cassava.

Symplasmic phloem loading and subcellular transport in storage roots are key factors for carbon allocation in cassava.

Cassava (Manihot esculenta) is a deciduous woody perennial shrub that stores large amounts of carbon and water in its storage roots. Previous studies have shown that assimilating unloading into storage roots happens symplasmically once secondary anatomy is established. However, mechanisms controlling phloem loading and overall carbon partitioning to different cassava tissues remain unclear. Here, we used a combination of histological, transcriptional, and biochemical analyses on different cassava tissues and at different timepoints to better understand source-sink carbon allocation. We found that cassava likely utilizes a predominantly passive symplasmic phloem loading strategy, indicated by the lack of expression of genes coding for key players of sucrose transport, the existence of branched plasmodesmata in the companion cell/bundle sheath interface of minor leaf veins, and very high leaf sucrose concentrations. Furthermore, we showed that tissue-specific changes in anatomy and non-structural carbohydrate contents are associated with tissue-specific modification in gene expression for sucrose cleavage/synthesis, as well as subcellular compartmentalization of sugars. Overall, our data suggest that carbon allocation during storage root filling is mostly facilitated symplasmically and is likely mostly regulated by local tissue demand and subcellular compartmentalization.

Proteomics of isolated sieve tubes from Nicotiana tabacum: sieve element-specific proteins reveal differentiation of the endomembrane system.

Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins ( SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.

Efficient sugar utilization and transition from oxidative to substrate-level phosphorylation in high starch storage roots of African cassava genotypes.

Cassava's storage roots represent one of the most important sources of nutritional carbohydrates worldwide. Particularly, smallholder farmers in sub-Saharan Africa depend on this crop plant, where resilient and yield-improved varieties are of vital importance to support steadily increasing populations. Aided by a growing understanding of the plant's metabolism and physiology, targeted improvement concepts already led to visible gains in recent years. To expand our knowledge and to contribute to these successes, we investigated storage roots of eight cassava genotypes with differential dry matter content from three successive field trials for their proteomic and metabolic profiles. At large, the metabolic focus in storage roots transitioned from cellular growth processes toward carbohydrate and nitrogen storage with increasing dry matter content. This is reflected in higher abundance of proteins related to nucleotide synthesis, protein turnover, and vacuolar energization in low starch genotypes, while proteins involved in sugar conversion and glycolysis were more prevalent in high dry matter genotypes. This shift in metabolic orientation was underlined by a clear transition from oxidative- to substrate-level phosphorylation in high dry matter genotypes. Our analyses highlight metabolic patterns that are consistently and quantitatively associated with high dry matter accumulation in cassava storage roots, providing fundamental understanding of cassava's metabolism as well as a data resource for targeted genetic improvement.

Analysis of companion cell and phloem metabolism using a transcriptome-guided model of Arabidopsis metabolism.

Companion cells and sieve elements play an essential role in vascular plants, and yet the details of the metabolism that underpins their function remain largely unknown. Here, we construct a tissue-scale flux balance analysis (FBA) model to describe the metabolism of phloem loading in a mature Arabidopsis (Arabidopsis thaliana) leaf. We explore the potential metabolic interactions between mesophyll cells, companion cells, and sieve elements based on the current understanding of the physiology of phloem tissue and through the use of cell type-specific transcriptome data as a weighting in our model. We find that companion cell chloroplasts likely play a very different role to mesophyll chloroplasts. Our model suggests that, rather than carbon capture, the most crucial function of companion cell chloroplasts is to provide photosynthetically generated ATP to the cytosol. Additionally, our model predicts that the metabolites imported into the companion cell are not necessarily the same metabolites that are exported in phloem sap; phloem loading is more efficient if certain amino acids are synthesized in the phloem tissue. Surprisingly, in our model predictions, the proton-pumping pyrophosphatase (H+-PPiase) is a more efficient contributor to the energization of the companion cell plasma membrane than the H+-ATPase.

Vernalization Alters Sink and Source Identities and Reverses Phloem Translocation from Taproots to Shoots in Sugar Beet.

During their first year of growth, overwintering biennial plants transport Suc through the phloem from photosynthetic source tissues to storage tissues. In their second year, they mobilize carbon from these storage tissues to fuel new growth and reproduction. However, both the mechanisms driving this shift and the link to reproductive growth remain unclear. During vegetative growth, biennial sugar beet (Beta vulgaris) maintains a steep Suc concentration gradient between the shoot (source) and the taproot (sink). To shift from vegetative to generative growth, they require a chilling phase known as vernalization. We studied sugar beet sink-source dynamics upon vernalization and showed that before flowering, the taproot underwent a reversal from a sink to a source of carbohydrates. This transition was induced by transcriptomic and functional reprogramming of sugar beet tissue, resulting in a reversal of flux direction in the phloem. In this transition, the vacuolar Suc importers and exporters TONOPLAST SUGAR TRANSPORTER2;1 and SUCROSE TRANSPORTER4 were oppositely regulated, leading to the mobilization of sugars from taproot storage vacuoles. Concomitant changes in the expression of floral regulator genes suggest that these processes are a prerequisite for bolting. Our data will help both to dissect the metabolic and developmental triggers for bolting and to identify potential targets for genome editing and breeding.

Multi-omics data integration reveals link between epigenetic modifications and gene expression in sugar beet (Beta vulgaris subsp. vulgaris) in response to cold.

BACKGROUND: DNA methylation is thought to influence the expression of genes, especially in response to changing environmental conditions and developmental changes. Sugar beet (Beta vulgaris ssp. vulgaris), and other biennial or perennial plants are inevitably exposed to fluctuating temperatures throughout their lifecycle and might even require such stimulus to acquire floral competence. Therefore, plants such as beets, need to fine-tune their epigenetic makeup to ensure phenotypic plasticity towards changing environmental conditions while at the same time steering essential developmental processes. Different crop species may show opposing reactions towards the same abiotic stress, or, vice versa, identical species may respond differently depending on the specific kind of stress. RESULTS: In this study, we investigated common effects of cold treatment on genome-wide DNA methylation and gene expression of two Beta vulgaris accessions via multi-omics data analysis. Cold exposure resulted in a pronounced reduction of DNA methylation levels, which particularly affected methylation in CHH context (and to a lesser extent CHG) and was accompanied by transcriptional downregulation of the chromomethyltransferase CMT2 and strong upregulation of several genes mediating active DNA demethylation. CONCLUSION: Integration of methylomic and transcriptomic data revealed that, rather than methylation having directly influenced expression, epigenetic modifications correlated with changes in expression of known players involved in DNA (de)methylation. In particular, cold triggered upregulation of genes putatively contributing to DNA demethylation via the ROS1 pathway. Our observations suggest that these transcriptional responses precede the cold-induced global DNA-hypomethylation in non-CpG, preparing beets for additional transcriptional alterations necessary for adapting to upcoming environmental changes.

High non-photochemical quenching of VPZ transgenic potato plants limits CO2 assimilation under high light conditions and reduces tuber yield under fluctuating light.

Under natural conditions, photosynthesis has to be adjusted to fluctuating light intensities. Leaves exposed to high light dissipate excess light energy in form of heat at photosystem II (PSII) by a process called non-photochemical quenching (NPQ). Upon fast transition from light to shade, plants lose light energy by a relatively slow relaxation from photoprotection. Combined overexpression of violaxanthin de-epoxidase (VDE), PSII subunit S (PsbS) and zeaxanthin epoxidase (ZEP) in tobacco accelerates relaxation from photoprotection, and increases photosynthetic productivity. In Arabidopsis, expression of the same three genes (VPZ) resulted in a more rapid photoprotection but growth of the transgenic plants was impaired. Here we report on VPZ expressing potato plants grown under various light regimes. Similar to tobacco and Arabidopsis, induction and relaxation of NPQ was accelerated under all growth conditions tested, but did not cause an overall increased photosynthetic rate or growth of transgenic plants. Tuber yield of VPZ expressing plants was unaltered as compared to control plants under constant light conditions and even decreased under fluctuating light conditions. Under control conditions, levels of the phytohormone abscisic acid (ABA) were found to be elevated, indicating an increased violaxanthin availability in VPZ plants. However, the increased basal ABA levels did not improve drought tolerance of VPZ transgenic potato plants under greenhouse conditions. The failure to benefit from improved photoprotection is most likely caused by a reduced radiation use efficiency under high light conditions resulting from a too strong NPQ induction. Mitigating this negative effect in the future might help to improve photosynthetic performance in VPZ expressing potato plants.

Metabolomics should be deployed in the identification and characterization of gene-edited crops.

Gene-editing techniques are currently revolutionizing biology, allowing far greater precision than previous mutagenic and transgenic approaches. They are becoming applicable to a wide range of plant species and biological processes. Gene editing can rapidly improve a range of crop traits, including disease resistance, abiotic stress tolerance, yield, nutritional quality and additional consumer traits. Unlike transgenic approaches, however, it is not facile to forensically detect gene-editing events at the molecular level, as no foreign DNA exists in the elite line. These limitations in molecular detection approaches are likely to focus more attention on the products generated from the technology than on the process in itself. Rapid advances in sequencing and genome assembly increasingly facilitate genome sequencing as a means of characterizing new varieties generated by gene-editing techniques. Nevertheless, subtle edits such as single base changes or small deletions may be difficult to distinguish from normal variation within a genotype. Given these emerging scenarios, downstream 'omics' technologies reflective of edited affects, such as metabolomics, need to be used in a more prominent manner to fully assess compositional changes in novel foodstuffs. To achieve this goal, metabolomics or 'non-targeted metabolite analysis' needs to make significant advances to deliver greater representation across the metabolome. With the emergence of new edited crop varieties, we advocate: (i) concerted efforts in the advancement of 'omics' technologies, such as metabolomics, and (ii) an effort to redress the use of the technology in the regulatory assessment for metabolically engineered biotech crops.

The Cassava Source-Sink project: opportunities and challenges for crop improvement by metabolic engineering.

Cassava (Manihot esculenta Crantz) is one of the important staple foods in Sub-Saharan Africa. It produces starchy storage roots that provide food and income for several hundred million people, mainly in tropical agriculture zones. Increasing cassava storage root and starch yield is one of the major breeding targets with respect to securing the future food supply for the growing population of Sub-Saharan Africa. The Cassava Source-Sink (CASS) project aims to increase cassava storage root and starch yield by strategically integrating approaches from different disciplines. We present our perspective and progress on cassava as an applied research organism and provide insight into the CASS strategy, which can serve as a blueprint for the improvement of other root and tuber crops. Extensive profiling of different field-grown cassava genotypes generates information for leaf, phloem, and root metabolic and physiological processes that are relevant for biotechnological improvements. A multi-national pipeline for genetic engineering of cassava plants covers all steps from gene discovery, cloning, transformation, molecular and biochemical characterization, confined field trials, and phenotyping of the seasonal dynamics of shoot traits under field conditions. Together, the CASS project generates comprehensive data to facilitate conventional breeding strategies for high-yielding cassava genotypes. It also builds the foundation for genome-scale metabolic modelling aiming to predict targets and bottlenecks in metabolic pathways. This information is used to engineer cassava genotypes with improved source-sink relations and increased yield potential.

Metabolic profiles of six African cultivars of cassava (Manihot esculenta Crantz) highlight bottlenecks of root yield.

Cassava is an important staple crop in sub-Saharan Africa, due to its high productivity even on nutrient poor soils. The metabolic characteristics underlying this high productivity are poorly understood including the mode of photosynthesis, reasons for the high rate of photosynthesis, the extent of source/sink limitation, the impact of environment, and the extent of variation between cultivars. Six commercial African cassava cultivars were grown in a greenhouse in Erlangen, Germany, and in the field in Ibadan, Nigeria. Source leaves, sink leaves, stems and storage roots were harvested during storage root bulking and analyzed for sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, protein, activities of enzymes in central metabolism and yield traits. High ratios of RuBisCO:phosphoenolpyruvate carboxylase activity support a C3 mode of photosynthesis. The high rate of photosynthesis is likely to be attributed to high activities of enzymes in the Calvin-Benson cycle and pathways for sucrose and starch synthesis. Nevertheless, source limitation is indicated because root yield traits correlated with metabolic traits in leaves rather than in the stem or storage roots. This situation was especially so in greenhouse-grown plants, where irradiance will have been low. In the field, plants produced more storage roots. This was associated with higher AGPase activity and lower sucrose in the roots, indicating that feedforward loops enhanced sink capacity in the high light and low nitrogen environment in the field. Overall, these results indicated that carbon assimilation rate, the K battery, root starch synthesis, trehalose, and chlorogenic acid accumulation are potential target traits for genetic improvement.

Auxin signaling and vascular cambium formation enable storage metabolism in cassava tuberous roots.

Cassava storage roots are among the most important root crops worldwide, and represent one of the most consumed staple foods in sub-Saharan Africa. The vegetatively propagated tropical shrub can form many starchy tuberous roots from its stem. These storage roots are formed through the activation of secondary root growth processes. However, the underlying genetic regulation of storage root development is largely unknown. Here we report distinct structural and transcriptional changes occurring during the early phases of storage root development. A pronounced increase in auxin-related transcripts and the transcriptional activation of secondary growth factors, as well as a decrease in gibberellin-related transcripts were observed during the early stages of secondary root growth. This was accompanied by increased cell wall biosynthesis, most notably increased during the initial xylem expansion within the root vasculature. Starch storage metabolism was activated only after the formation of the vascular cambium. The formation of non-lignified xylem parenchyma cells and the activation of starch storage metabolism coincided with increased expression of the KNOX/BEL genes KNAT1, PENNYWISE, and POUND-FOOLISH, indicating their importance for proper xylem parenchyma function.

Transcriptional and Metabolic Profiling of Potato Plants Expressing a Plastid-Targeted Electron Shuttle Reveal Modulation of Genes Associated to Drought Tolerance by Chloroplast Redox Poise.

Water limitation represents the main environmental constraint affecting crop yield worldwide. Photosynthesis is a primary drought target, resulting in over-reduction of the photosynthetic electron transport chain and increased production of reactive oxygen species in plastids. Manipulation of chloroplast electron distribution by introducing alternative electron transport sinks has been shown to increase plant tolerance to multiple environmental challenges including hydric stress, suggesting that a similar strategy could be used to improve drought tolerance in crops. We show herein that the expression of the cyanobacterial electron shuttle flavodoxin in potato chloroplasts protected photosynthetic activities even at a pre-symptomatic stage of drought. Transcriptional and metabolic profiling revealed an attenuated response to the adverse condition in flavodoxin-expressing plants, correlating with their increased stress tolerance. Interestingly, 5-6% of leaf-expressed genes were affected by flavodoxin in the absence of drought, representing pathways modulated by chloroplast redox status during normal growth. About 300 of these genes potentially contribute to stress acclimation as their modulation by flavodoxin proceeds in the same direction as their drought response in wild-type plants. Tuber yield losses under chronic water limitation were mitigated in flavodoxin-expressing plants, indicating that the flavoprotein has the potential to improve major agronomic traits in potato.

Symplasmic phloem unloading and radial post-phloem transport via vascular rays in tuberous roots of Manihot esculenta.

Cassava (Manihot esculenta) is one of the most important staple food crops worldwide. Its starchy tuberous roots supply over 800 million people with carbohydrates. Yet, surprisingly little is known about the processes involved in filling of those vital storage organs. A better understanding of cassava carbohydrate allocation and starch storage is key to improving storage root yield. Here, we studied cassava morphology and phloem sap flow from source to sink using transgenic pAtSUC2::GFP plants, the phloem tracers esculin and 5(6)-carboxyfluorescein diacetate, as well as several staining techniques. We show that cassava performs apoplasmic phloem loading in source leaves and symplasmic unloading into phloem parenchyma cells of tuberous roots. We demonstrate that vascular rays play an important role in radial transport from the phloem to xylem parenchyma cells in tuberous roots. Furthermore, enzymatic and proteomic measurements of storage root tissues confirmed high abundance and activity of enzymes involved in the sucrose synthase-mediated pathway and indicated that starch is stored most efficiently in the outer xylem layers of tuberous roots. Our findings form the basis for biotechnological approaches aimed at improved phloem loading and enhanced carbohydrate allocation and storage in order to increase tuberous root yield of cassava.

Differences and commonalities of plant responses to single and combined stresses.

In natural or agricultural environments, plants are constantly exposed to a wide range of biotic and abiotic stresses. Given the forecasted global climate changes, plants will cope with heat waves, drought periods and pathogens at the same time or consecutively. Heat and drought cause opposing physiological responses, while pathogens may or may not profit from climate changes depending on their lifestyle. Several studies have been conducted to find stress-specific signatures or stress-independent commonalities. Previously this has been done by comparing different single stress treatments. This approach has been proven difficult since most studies, comparing single and combined stress conditions, have come to the conclusion that each stress treatment results in specific transcriptional changes. Although transcriptional changes at the level of individual genes are highly variable and stress-specific, central metabolic and signaling responses seem to be common, often leading to an overall reduced plant growth. Understanding how specific transcriptional changes are linked to stress adaptations and identifying central hubs controlling this interaction will be the challenge for the coming years. In this review, we will summarize current knowledge on plant responses to different individual and combined stresses and try to find a common thread potentially underlying these responses. We will begin with a brief summary of known physiological, metabolic, transcriptional and hormonal responses to individual stresses, elucidate potential commonalities and conflicts and finally we will describe results obtained during combined stress experiments. Here we will concentrate on simultaneous application of stress conditions but we will also touch consequences of sequential stress treatments.

Comparative proteomic profiling of the choline transporter-like1 (CHER1) mutant provides insights into plasmodesmata composition of fully developed Arabidopsis thaliana leaves.

In plants, intercellular communication and exchange are highly dependent on cell wall bridging structures between adhering cells, so-called plasmodesmata (PD). In our previous genetic screen for PD-deficient Arabidopsis mutants, we described choline transporter-like 1 (CHER1) being important for PD genesis and maturation. Leaves of cher1 mutant plants have up to 10 times less PD, which do not develop to complex structures. Here we utilize the T-DNA insertion mutant cher1-4 and report a deep comparative proteomic workflow for the identification of cell-wall-embedded PD-associated proteins. Analyzing triplicates of cell-wall-enriched fractions in depth by fractionation and quantitative high-resolution mass spectrometry, we compared > 5000 proteins obtained from fully developed leaves. Comparative data analysis and subsequent filtering generated a list of 61 proteins being significantly more abundant in Col-0. This list was enriched for previously described PD-associated proteins. To validate PD association of so far uncharacterized proteins, subcellular localization analyses were carried out by confocal laser-scanning microscopy. This study confirmed the association of PD for three out of four selected candidates, indicating that the comparative approach indeed allowed identification of so far undescribed PD-associated proteins. Performing comparative cell wall proteomics of Nicotiana benthamiana tissue, we observed an increase in abundance of these three selected candidates during sink to source transition. Taken together, our comparative proteomic approach revealed a valuable data set of potential PD-associated proteins, which can be used as a resource to unravel the molecular composition of complex PD and to investigate their function in cell-to-cell communication.

Choline transporter-like1 (CHER1) is crucial for plasmodesmata maturation in Arabidopsis thaliana.

Plasmodesmata (PD) are microscopic pores connecting plant cells and enable cell-to-cell transport. Currently, little information is known about the molecular mechanisms regulating PD formation and development. To uncover components of PD development we made use of the 17 kDa movement protein (MP17) encoded by the Potato leafroll virus (PLRV). The protein is required for cell-to-cell movement of the virus and localises to complex PD. Forward genetic screening for Arabidopsis mutants with altered PD binding of MP17 revealed several mutant lines, while molecular genetics, biochemical and microscopic studies allowed further characterisation. Map-based cloning of one mutant revealed a point mutation in the choline transporter-like 1 (CHER1) protein, changing glycine247 into glutamate. Mutation in CHER1 resulted in a starch excess phenotype and stunted growth. Ultrastructure analysis of shoot apical meristems, developing and fully developed leaves showed reduced PD numbers and the absence of complex PD in fully developed leaves. This indicates that cher1 mutants are impaired in PD formation and development. Global lipid profiling revealed only slight modifications in the overall lipid composition, however, altered composition of PD-associated lipids cannot be ruled out. Thus, cher1 is devoid of complex PD in developed leaves and provides insights into the formation of complex PD at the molecular level.

Deciphering source and sink responses of potato plants (Solanum tuberosum L.) to elevated temperatures.

Potato is an important staple food with increasing popularity worldwide. Elevated temperatures significantly impair tuber yield and quality. Breeding heat-tolerant cultivars is therefore an urgent need to ensure sustainable potato production in the future. An integrated approach combining physiology, biochemistry, and molecular biology was undertaken to contribute to a better understanding of heat effects on source- (leaves) and sink-organs (tubers) in a heat-susceptible cultivar. An experimental set-up was designed allowing tissue-specific heat application. Elevated day and night (29°C/27°C) temperatures impaired photosynthesis and assimilate production. Biomass allocation shifted away from tubers towards leaves indicating reduced sink strength of developing tubers. Reduced sink strength of tubers was paralleled by decreased sucrose synthase activity and expression under elevated temperatures. Heat-mediated inhibition of tuber growth coincided with a decreased expression of the phloem-mobile tuberization signal SP6A in leaves. SP6A expression and photosynthesis were also affected, when only the belowground space was heated, and leaves were kept under control conditions. By contrast, the negative effects on tuber metabolism were attenuated, when only the shoot was subjected to elevated temperatures. This, together with transcriptional changes discussed, indicated a bidirectional communication between leaves and tubers to adjust the source capacity and/or sink strength to environmental conditions.