Biology subjects

Berkowitz, O.

Publications and source records attributed to Berkowitz, O..

10 recordsLinked to original sources

bioRxiv2025

METABOLIC AND TRANSCRIPTOMIC ANALYSES IDENTIFY COORDINATED RESOURCE REALLOCATION IN RESPONSE TO PHOSPHATE SUPPLY IN HEMP

Cannabis sativa L., one of the earliest domesticated crops, was selectively bred into drug-type (high phytocannabinoid yield) and hemp-type (fibre and seed production) varieties, resulting in divergent traits including nutrient requirements. To address the limited molecular understanding of nutrient use regulation in Cannabis, this study investigated the response of dual-purpose hemp to varying phosphate availability. Physiological and transcriptomic profiling across source and sink organs revealed that biomass partitioning to flowers was sustained unless phosphate was completely withdrawn. Analyses of key phosphate-responsive genes highlighted the importance of P reallocation to flowers via glycolytic bypass reactions and release of phosphate from organic pools in source organs. Phospholipid biosynthesis and other P-dependent metabolic pathways remained active in sink organs. While the transcriptional response of key regulators, such as SPX DOMAIN GENE (SPX), PHOSPHATE STARVATION RESPONSE1 (PHR1) and NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIONAL REPRESSOR1 (NIGT1) followed canonical patterns, our data suggest a lack of suppression of phosphate uptake and root-to-shoot translocation at higher phosphate supply levels. Subsequent depletion of nitrate pools restricts further growth. These findings uncover conserved and unique aspects of nutrient signalling in hemp, shedding light on its adaptation to nutrient-poor soils through distinct source-sink regulation. HIGHLIGHTContrasting human selection in Cannabis sativa enables the analysis of divergent nutrient-use strategies. Our integrated transcriptomic and physiological data revealed adaptive resource allocation, informing the development of nutrient-efficient cultivars.

plant biology↗

bioRxiv2023

FTSH3 facilitates complex I degradation through a direct interaction with the complex I subunit PSST

Complex I (NADH dehydrogenase), the largest complex involved in mitochondrial oxidative phosphorylation is composed of nuclear and mitochondrial encoded subunits. Its assembly requires sequential addition of subdomains and modules. As it is prone to oxidative damage, complex I subunits continually undergo proteolysis and turnover. We describe the mechanism by which complex I abundance is regulated in a complex I deficient mutant. Using a forward genetic approach we have identified that the complex I Q-module domain subunit PSST, interacts with FTSH3 (Filamentous Temperature Sensitive H3) to mediate the disassembly of the matrix arm domain module for proteolysis and turnover as a means of protein quality control. We show the direct interaction of FTSH3 with PSST and identify the amino acid residues required for this interaction. It is the ATPase function of FTSH3 that is required for the interaction, as the mutation can be compensated by a proteolytically inactive form of FTSH3. Furthermore, it cannot be compensated by FTSH10 that is also located in mitochondria, as the latter does not interact with PSST. This study reveals the mechanistic process at the resolution of the residues involved of how FTSH3 recognises complex I for degradation.

plant biology↗

bioRxiv2023

Spatially Resolved Transcriptomic Analysis of the Germinating Barley Grain

Seeds, which provide a major source of calories for humans, are a unique stage of a flowering plant s lifecycle. During seed germination the embryo reactivates rapidly and goes through major developmental transitions to become a seedling. This requires extensive and complex spatiotemporal coordination of cell and tissue activity. Existing gene expression profiling methods, such as laser capture microdissection followed by RNA-seq and single-cell RNA-seq, suffer from either low throughput or the loss of spatial information about the cells analysed. Spatial transcriptomics methods couple high throughput analysis of gene expression simultaneously with the ability to record the spatial location of each individual region analysed. We developed a spatial transcriptomics workflow for germinating barley grain to better understand the spatiotemporal control of gene expression within individual seed cell types. More than 14,000 genes were differentially regulated across 0, 1, 3, 6 and 24 hours after imbibition. This approach enabled us to observe that many functional categories displayed specific spatial expression patterns that could be resolved at a sub-tissue level. Individual aquaporin gene family members, important for water and ion transport, had specific spatial expression patterns over time, as well as genes related to cell wall modification, membrane transport and transcription factors. Using spatial autocorrelation algorithms, we were able to identify auxin transport genes that had increasingly focused expression within subdomains of the embryo over germination time, suggestive of a role in establishment of the embryo axis. Together, our data provides an unprecedented spatially resolved cellular map for barley grain germination and specific genes to target for functional genomics to define cellular restricted processes in tissues during germination. The data can be viewed at https://spatial.latrobe.edu.au/.

plant biology↗

bioRxiv2021

Single Cell App: An App for Single Cell RNA-sequencing Data Visualization, Comparison and Discovery

The Single Cell App is a cloud-based application that allows visualisation and comparison of scRNA-seq data and is scalable according to use. Users upload their own or publicly available scRNA-seq datasets after pre-processing to be visualised using a web browser. The data can be viewed in two colour modes, Cluster - representing cell identity, and Values - level of expression, and data queried using keyword or gene identification number(s). Using the app to compare four different studies we determined that some genes frequently used as cell-type markers are in fact study specific. Phosphate transporter and hormone response genes were exemplary investigated with the app. This showed that the apparent cell specific expression of PHO1;H3 differed between GFP-tagging and scRNA-seq studies. Some phosphate transporter genes were induced by protoplasting, they retained cell specificity, indicating that cell specific stress responses (i.e. protoplasting). Examination of the cell specificity of hormone response genes revealed that 132 hormone responsive genes display restricted expression and that the jasmonate response gene TIFY8 is expressed in endodermal cells which differs from previous reports. It also appears that JAZ repressors have cell-type specific functions. These differences, identified using the Single Cell App, highlight the need for resources to enable researchers to find common and different patterns of cell specific gene expression. Thus, the Single Cell App enables researchers to form new hypothesis, perform comparative studies, allows for easy re-use of data for this emerging technology to provide novel avenues to crop improvement.

plant biology↗

bioRxiv2021

GmSALT3 expression improves reactive oxygen species detoxification in salt-stressed soybean roots

Soybean plants are salinity (NaCl) sensitive, with their yield significantly decreased under moderately saline conditions. GmSALT3 is the dominant gene underlying a major QTL for salt tolerance in soybean. GmSALT3 encodes a transmembrane protein belonging to the plant cation/proton exchanger (CHX) family. It is currently unknown through which molecular mechanism(s) the ER-localised GmSALT3 contributes to salinity tolerance, as its localisation excludes direct involvement in ion exclusion. In order to gain insights into potential molecular mechanism(s), we used RNA-seq analysis of roots from two soybean NILs (Near Isogenic Lines); NIL-S (salt-sensitive, Gmsalt3) and NIL-T (salt-tolerant, GmSALT3), grown under control and saline conditions (200 mM NaCl) at three time points (0h, 6h, and 3 days). Gene ontology (GO) analysis showed that NIL-T has greater responses aligned to oxidation reduction. ROS were shown less abundant and scavenging enzyme activity was higher in NIL-T, consistent with the RNA-seq data. Further analysis indicated that genes related to calcium signalling, vesicle trafficking and Casparian strip (CS) development were upregulated in NIL-T following salt treatment. We propose that GmSALT3 improves the ability of NIL-T to cope with saline stress through preventing ROS overaccumulation in roots, and potentially modulating Ca2+ signalling, vesicle trafficking and formation of diffusion barriers. HighlightRNA-seq analysis revealed that GmSALT3, which confers improved salt tolerance on soybean, improves reactive oxygen species detoxification in roots.

plant biology↗

bioRxiv2020

RNA-seq analysis of laser micro-dissected Arabidopsis thaliana leaf epidermis, mesophyll and vasculature defines tissue-specific transcriptional responses to multiple stress treatments

Acclimation of plants to adverse environmental conditions requires the coordination of gene expression and signalling pathways between tissues and cell types. As the energy and carbon capturing organs, leaves are significantly affected by abiotic and biotic stresses. However, tissue- or cell type-specific analyses of stress responses have largely focussed on the Arabidopsis root. Here, we comparatively explore the transcriptomes of three leaf tissues (epidermis, mesophyll, vasculature) after induction of diverse stress pathways by chemical stimuli (antimycin A, 3-amino-1,2,4-triazole, methyl viologen, salicylic acid) and UV light in Arabidopsis. Profiling stimuli-dependent changes after treatments revealed an overall reduction in the tissue-specific expression of genes, with only a limited number gaining or changing their tissue-specificity. We find no evidence of a common stress response, with only a few genes responsive to two or more treatments in the analysed tissues. However, differentially expressed genes overlap across tissues for individual treatments. Further analyses provided evidence for an interaction of auxin and ethylene that mediates retrograde signalling during mitochondrial dysfunction specifically in the epidermis, and a gene regulatory network defined the hierarchy of interactions. Taken together, we generated an extensive reference data set and results enable the tailoring of the tissue-specific engineering of stress tolerant plants.

plant biology↗

bioRxiv2020

Identification of a unique ZIP transporter involved in zinc uptake via the arbuscular mycorrhizal fungal pathway

Low soil zinc (Zn) availability is a limiting factor for crop yield, and increasing Zn content is a major target for the biofortification of major crops. Arbuscular mycorrhizal (AM) fungi associate with the roots of most terrestrial plant species and improve the host plants growth and nutrition through the mycorrhizal pathway of nutrient uptake. Although the physiology of Zn uptake through the mycorrhizal pathway is well established, the identity of the molecular components responsible for Zn transport in the mycorrhizal pathway are unknown. RNA-seq analysis identified the putative Zn transporter gene MtZIP14 by its marked up-regulation in Medicago truncatula roots when colonised by the AM fungus Rhizophagus irregularis under varying soil Zn supply. Expression of GFP-tagged MtZIP14 in roots revealed that it is exclusively localised to the site of plant-fungal nutrient exchange in cortical cells, the peri-arbuscular membrane. Expression of MtZIP14 in a yeast mutant lacking Zn transport function restored growth under low Zn availability. M. truncatula MtZIP14 loss-of-function mutants had reduced shoot biomass compared to the wild-type when colonised by AM fungi and grown under low Zn. Vesicular and arbuscular colonisation, but not hyphal colonisation, were also lower in mtzip14 mutant plants. Based on these results we propose that MtZIP14 plays a key role in the transport of Zn from AM fungus to plant across the peri-arbuscular membrane, and MtZIP14 function is crucial to plant competitiveness in a low Zn soil. Significance statementMajority of crop plant species associate with arbuscular mycorrhizal fungi, which can increase plant nutrient uptake. Improving our knowledge of how Zn is taken up in mycorrhizal plants will lead to improved plant and human Zn nutrition outcomes. Here, we report a novel plant transporter with a major role in Zn nutrition of mycorrhizal plants. MtZIP14 is involved in Zn transport, is exclusively localised to the specialised plant-fungal interface in roots, and impairment of MtZIP14 gene function results in negative impacts on both plant growth and Zn nutrition.

plant biology↗

bioRxiv2020

The genetic origin of evolidine, the first cyclopeptide discovered in plants, and related orbitides

Cyclic peptides are reported to have antibacterial, antifungal and other bioactivities. Several genera of the Rutaceae family are known to produce orbitides, which are small head-to-tail cyclic peptides composed of proteinogenic amino acids and lacking disulfide bonds. Melicope xanthoxyloides is an Australian rain forest tree of the Rutaceae family in which evolidine - the first plant cyclic peptide - was discovered. Evolidine (cyclo-SFLPVNL) has subsequently been all but forgotten in the academic literature, but here we use tandem mass spectrometry to rediscover evolidine and using de novo transcriptomics we show its biosynthetic origin to be from a short precursor just 48 residues in length. In all, seven M. xanthoxyloides orbitides were found and they had atypically diverse C-termini consisting of residues not recognized by either of the known proteases plants use to macrocyclize peptides. Two of the novel orbitides were studied by nuclear magnetic resonance spectroscopy and although one had definable structure, the other did not. By mining RNA-seq and whole genome sequencing data from other species, it was apparent that a large and diverse family of peptides is encoded by sequences like these across the Rutaceae.

biochemistry↗

bioRxiv2020

Developmental Normalization of Phenomics Data Generated by High Throughput Plant Phenotyping Systems

BackgroundSowing time is commonly used as the temporal reference for Arabidopsis thaliana (Arabidopsis) experiments in high throughput plant phenotyping (HTPP) systems. This relies on the assumption that germination and seedling establishment are uniform across the population. However, individual seeds have different development trajectories even under uniform environmental conditions. This leads to increased variance in quantitative phenotyping approaches. We developed the Digital Adjustment of Plant Development (DAPD) normalization method. It normalizes time-series HTPP measurements by reference to an early developmental stage and in an automated manner. The timeline of each measurement series is shifted to a reference time. The normalization is determined by cross-correlation at multiple time points of the time-series measurements, which may include rosette area, leaf size, and number. ResultsThe DAPD method improved the accuracy of phenotyping measurements by decreasing the statistical dispersion of quantitative traits across a time-series. We applied DAPD to evaluate the relative growth rate in A. thaliana plants and demonstrated that it improves uniformity in measurements, permitting a more informative comparison between individuals. Application of DAPD decreased variance of phenotyping measurements by up to 2.5 times compared to sowing-time normalization. The DAPD method also identified more outliers than any other central tendency technique applied to the non-normalized dataset.

plant biology↗

bioRxiv2019

Novel cyclic peptides in seeds of Annona muricata are ribosomally synthesised

Small, cyclic peptides are reported to have many bioactivities. In bacteria and fungi they can be made by non-ribosomal peptide synthetases, but in plants they are exclusively ribosomal. Cyclic peptides from the Annona genus possess cytotoxic and anti-inflammatory activities, but their biosynthesis is unknown. The medicinal soursop plant, Annona muricata, contains annomuricatins A (cyclo-PGFVSA) and B (cyclo-PNAWLGT). Here, using de novo transcriptomics and tandem mass spectrometry, we identify a suite of short transcripts for precursor proteins for ten validated annomuricatins, nine of which are novel. In their precursors, annomuricatins are preceded by an absolutely conserved Glu and each peptide sequence has a conserved proto-C-terminal Pro, revealing parallels with the segetalin orbitides from the seed of Vaccaria hispanica, which are processed through ligation by a prolyl oligopeptidase in a transpeptidation reaction.

biochemistry↗