Biology subjects

Weber, A. P.

Publications and source records attributed to Weber, A. P..

5 recordsLinked to original sources

bioRxiv2024

Single-nuclei sequencing of Moricandia arvensis reveals bundle sheath cell function in the photorespiratory shuttle of C3-C4 intermediate Brassicaceae

Spatially confined gene expression determines cell identity and is fundamental to complex plant traits. In the evolutionary transition from C3 to the more efficient C4 photosynthesis, restricting the glycine decarboxylase reaction to bundle sheath cells initiates a carbon concentrating mechanism via the photorespiratory glycine shuttle. This evolutionary step is generally thought to play an essential role in the progression from ancestral C3 to C4 photosynthesis. Plants operating this shuttle are often referred to as C3-C4 intermediates or C2 species. Within the Brassicaceae family, which includes model plants and crops, such species have evolved independently at least five times. However, research on the biochemistry of C3-C4 intermediates in the Brassicaceae has been limited to a few case studies of differentially localized proteins between mesophyll and bundle sheath cells. Here, we leveraged recent advances in single-cell transcriptome sequencing to better understand how new cellular specialization affects interconnected pathways. We generated a single-nuclei RNA sequencing dataset for Moricandia arvensis, a Brassicaceae with C3-C4 intermediate characteristics, and compared it to a publicly available single-cell transcriptome of leaf tissue of the C3 Arabidopsis thaliana. We independently confirmed the localization of selected photorespiratory proteins by electron microscopy of immunogold-labelled leaf sections. Our analysis revealed the shift in expression of genes directly associated with the photorespiratory glycine decarboxylase reaction, but also of related pathways, such as ammonium assimilation, synthesis of specific amino acids, redox regulation, and transport to the M. arvensis bundle sheath. In contrast, the expression of these genes was not restricted to this cell type in the C3 plant. HighlightSingle-nucleus RNA sequencing of Moricandia arvensis reveals bundle sheath cell-specific expression of photorespiration genes and associated pathways beyond glycine decarboxylase, including ammonium assimilation and redox regulation. This highlights the key role of metabolic compartmentalization in supporting C3-C4 intermediate photosynthesis.

plant biology↗

bioRxiv2023

Transcription factors mediating regulation of photosynthesis

Photosynthesis by which plants convert carbon dioxide to sugars using the energy of light is fundamental to life as it forms the basis of nearly all food chains. Surprisingly, our knowledge about its transcriptional regulation remains incomplete. Effort for its agricultural optimization have mostly focused on post-translational regulatory processes1-3 but photosynthesis is regulated at the post-transcriptional4 and the transcriptional level5. Stacked transcription factor mutations remain photosynthetically active5,6 and additional transcription factors have been difficult to identify possibly due to redundancy6 or lethality. Using a random forest decision tree-based machine learning approach for gene regulatory network calculation7 we determined ranked candidate transcription factors and validated five out of five tested transcription factors as controlling photosynthesis in vivo. The detailed analyses of previously published and newly identified transcription factors suggest that photosynthesis is transcriptionally regulated in a partitioned, non-hierarchical, interlooped network.

plant biology↗

bioRxiv2020

Overexpression of the chloroplastic 2-oxoglutarate/malate transporter in rice disturbs carbon and nitrogen homeostasis

The chloroplastic oxaloacetate/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of oxaloacetate (OAA). Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing -ketoglutarate for the GS/GOGAT reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C4 photosynthesis. OAA resulting from PEP-carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-MDH, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. Therefore, to engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp. indica IR64). Increased activity of the transporter in transgenic rice was confirmed by reconstitution of transporter activity into proteoliposomes. Unexpectedly, over-expression of ZmOMT1 in rice negatively affected growth, CO2 assimilation rate, total free amino acid contents, TCA cycle metabolites, as well as sucrose and starch contents. Accumulation of high amounts of aspartate and the impaired growth phenotype of OMT1 rice lines could be suppressed by simultaneous over-expression of ZmDiT2. Implications for engineering C4-rice are discussed.

plant biology↗

bioRxiv2019

Rapid single-step affinity purification of HA-tagged mitochondria from Arabidopsis thaliana

Photosynthesis in plant cells would not be possible without the supportive role of mitochondria. However, isolation of mitochondria from plant cells, for physiological and biochemical analyses, is a lengthy and tedious process. Established isolation protocols require multiple centrifugation steps and substantial amounts of starting material. To overcome these limitations, we tagged mitochondria in Arabidopsis thaliana with a triple haemagglutinin-tag for rapid purification via a single affinity purification step. This protocol yields a substantial quantity of highly pure mitochondria from 1 g of Arabidopsis seedlings. The purified mitochondria were suitable for enzyme activity analyses and yielded sufficient amounts of proteins for deep proteomic profiling. We applied this method for the proteomic analysis of the Arabidopsis bou-2 mutant deficient in the mitochondrial glutamate transporter A bout de souffle (BOU) and identified 27 differentially expressed mitochondrial proteins compared with transgenic Col-0 controls. Our work also sets the stage for the development of advanced mitochondria isolation protocols for distinct cell types.\n\nOne-sentence summaryAffinity-tagging of mitochondria in plant cells with a triple hemagglutinin-tag enables single-step affinity purification of mitochondria in less than 20 min.

plant biology↗

bioRxiv2018

Temperature but not the circadian clock determines nocturnal carbohydrate availability for growth in cereals

The circadian clock is considered a key target for crop improvement because it controls metabolism and growth in Arabidopsis. Here, we show that the clock gene EARLY FLOWERING 3 (ELF3) controls vegetative growth in Arabidopsis but not in the cereal crop barley. Growth in Arabidopsis is determined by the degradation of leaf starch reserves at night, which is controlled by ELF3. The vegetative growth of barley, however, is determined by the depletion of leaf sucrose stores through an exponential kinetics, presumably catalyzed by the vacuolar sucrose exporter SUCROSE TRANSPORTER 2 (SUT2). This process depends on the sucrose content and the nighttime temperature but not on ELF3. The regulation of starch degradation and sucrose depletion in barley ensures efficient growth at favorable temperature as stores become exhausted at dawn. On cool nights, however, only the starch degradation rate is compensated against low nighttime temperatures, whereas the sucrose depletion rate is reduced. This coincides with reduced biomass in barley but not in Arabidopsis after growth in consecutive cool nights. The sucrose depletion metabolism determines growth in the cereal crops barley, wheat, and rice but is not generally conserved in monocot species and is not a domestication-related trait. Therefore, the control of growth by endogenous (clock) versus external factors (temperature) is species-specific and depends on the predominant carbohydrate store. Our results give new insights into the physiology of growth in cereals and provide a basis for studying the genetics and evolution of different carbohydrate stores and their contribution to plant productivity and adaptation.\n\nSignificance StatementThe circadian clock controls growth in the model plant Arabidopsis thaliana by regulating the starch degradation rate so that reserves last until dawn. This prevents nocturnal starvation until photosynthesis resumes. The cereal crops barley, wheat and rice, however, predominantly consume sucrose instead of starch as carbohydrate source. We find that carbohydrate supply from sucrose at night is regulated by enzyme kinetics and night-time temperature, but not the circadian clock. We postulate that the regulation of growth depends on the predominant carbohydrate store, where starch degradation is controlled by endogenous cues (clock) and sucrose depletion by external factors (temperature). These differences in the regulation of carbohydrate availability at night may have important implications for adapting crops yields to climate change.

plant biology↗