Biology subjects

Hibberd, J. M.

Publications and source records attributed to Hibberd, J. M..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

An optimised transformation protocol for Anthoceros agrestis and three more hornwort species

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimised transformation protocol for A. agrestis which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussi and Phaeoceros carolinianus. The new transformation method is less laborious, faster and results in the generation of greatly increased numbers of transformants compared to the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localisation signal peptides for hornworts providing new tools to better understand hornwort cell biology.

plant biology↗

bioRxiv2022

The genome of Gynandropsis gynandra provides insights into whole-genome duplications and the evolution of C4 photosynthesis in Cleomaceae

Gynandropsis gynandra (Cleomaceae) is a cosmopolitan leafy vegetable and medicinal plant, which has also been used as a model to study C4 photosynthesis due to its evolutionary proximity to Arabidopsis. Here, we present a high-quality genome sequence of G. gynandra, anchored onto 17 main super- scaffolds with a total length of 740 Mb, an N50 of 42 Mb and 30,933 well-supported gene models. The G. gynandra genome and previously released genomes of C3 relatives in the Cleomaceae and Brassicaceae make an excellent model for studying the role of genome evolution in the transition from C3 to C4 photosynthesis. We revealed that G. gynandra and its C3 relative Tarenaya hassleriana shared a whole-genome duplication event (Gg-), then an addition of a third genome (Th-, +1x) took place in T. hassleriana but not in G. gynandra. Analysis of syntenic copy number of C4 photosynthesis-related gene families indicates that G. gynandra generally retained more duplicated copies of these genes than C3 T. hassleriana, and also that the G. gynandra C4 genes might have been under positive selection pressure. Both whole-genome and single-gene duplication were found to contribute to the expansion of the aforementioned gene families in G. gynandra. Collectively, this study enhances our understanding of the impact of gene duplication and gene retention on the evolution of C4 photosynthesis in Cleomaceae.

plant biology↗

bioRxiv2022

Abscisic Acid modulates neighbor proximity-induced leaf hyponasty in Arabidopsis

Leaves of shade avoiding plants like Arabidopsis thaliana change their growth pattern and position in response to a low red to far-red ratio (LRFR) that is encountered in dense plant communities. In LRFR, transcription factors of the phytochrome interacting family (PIFs) are de-repressed. PIFs induce auxin production, which is required to promote leaf hyponasty thereby favoring access to unfiltered sunlight. Abscisic acid (ABA) has also been implicated in the control of leaf hyponasty. In addition, gene expression patterns suggest that LRFR regulates the ABA response. Here we show that, in leaves, LRFR leads to a rapid increase in ABA levels. Changes in ABA levels depend on PIFs, which regulate the expression of genes encoding isoforms of the enzyme catalyzing a rate-limiting step in ABA biosynthesis. Interestingly, ABA biosynthesis and signaling mutants have more erect leaves in white light but respond less to LRFR. Consistent with this, ABA application decreases the leaf angle in white light but this response is inhibited in LRFR. Tissue-specific interference with ABA signaling indicates that an ABA response is required in different cell types for LRFR-induced hyponasty. Collectively, our data indicate LRFR triggers rapid PIF-mediated ABA production. ABA plays a different role in controlling hyponasty in white light compared to LRFR. Moreover, ABA exerts its activity in multiple cell types to control leaf position. One sentence summaryAbscisic acid biosynthesis and signaling are required to promote leaf hyponasty in response to a drop in the red to far-red ratio.

plant biology↗

bioRxiv2021

Phytochrome B regulates reactive oxygen signaling during abiotic and biotic stress in plants

Plants are essential for life on Earth converting light into chemical energy in the form of sugars. To adjust for changes in light intensity and quality, and to become as efficient as possible in harnessing light, plants utilize multiple light receptors, signaling, and acclimation mechanisms. In addition to altering plant metabolism, development and growth, light cues sensed by some photoreceptors, such as phytochromes, impact on many plant responses to biotic and abiotic stresses. Central for plant responses to different stresses are reactive oxygen species (ROS) that function as key signaling molecules. Recent studies demonstrated that respiratory burst oxidase homolog (RBOH) proteins that reside at the plasma membrane and produce ROS at the apoplast play a key role in plant responses to different biotic and abiotic stresses. Here we reveal that phytochrome B (phyB) and RBOHs function as part of a key regulatory module that controls ROS production, transcript expression, and plant acclimation to excess light stress. We further show that phyB can regulate ROS production during stress even if it is restricted to the cytosol, and that phyB, RBOHD and RBOHF co-regulate thousands of transcripts in response to light stress. Surprisingly, we found that phyB is also required for ROS accumulation in response to heat, wounding, cold, and bacterial infection. Taken together, our findings reveal that phyB plays a canonical role in plant responses to biotic and abiotic stresses, regulating ROS production, and that phyB and RBOHs function in the same pathway. Significant StatementAbiotic and biotic stresses cause extensive losses to agricultural production and threaten global food security. Augmenting plant resilience to stressful conditions requires understanding of how plants sense stress. Here we report that the sensing of different abiotic and biotic stresses that result in the production of the key stress-response signaling molecules, reactive oxygen species, requires the plant photoreceptor protein phytochrome B. We further show that in contrast to its many nuclear functions, phytochrome B regulates reactive oxygen production by plasma membrane-localized respiratory burst oxidase homologs while localized to the cytosol. Our findings reveal the existence of a rapid stress response regulatory mechanism requiring phytochrome B and reactive oxygen species, essential for plant acclimation to stress.

plant biology↗

bioRxiv2021

The bundle sheath of rice is conditioned to play an active role in water transport as well as sulfur assimilation and jasmonic acid synthesis

Leaves comprise multiple cell types but our knowledge of the patterns of gene expression that underpin their functional specialization is fragmentary. Our understanding and ability to undertake rational redesign of these cells is therefore limited. We aimed to identify genes associated with the incompletely understood bundle sheath of C3 plants, which represents a key target associated with engineering traits such as C4 photosynthesis into rice. To better understand veins, bundle sheath and mesophyll cells of rice we used laser capture microdissection followed by deep sequencing. Gene expression of the mesophyll is conditioned to allow coenzyme metabolism and redox homeostasis as well as photosynthesis. In contrast, the bundle sheath is specialized in water transport, sulphur assimilation and jasmonic acid biosynthesis. Despite the small chloroplast compartment of bundle sheath cells, substantial photosynthesis gene expression was detected. These patterns of gene expression were not associated with presence/absence of particular transcription factors in each cell type, but rather gradients in expression across the leaf. Comparative analysis with C3 Arabidopsis identified a small gene-set preferentially expressed in bundle sheath cells of both species. This included genes encoding transcription factors from fourteen orthogroups, and proteins allowing water transport, sulphate assimilation and jasmonic acid synthesis. The most parsimonious explanation for our findings is that bundle sheath cells from the last common ancestor of rice and Arabidopsis was specialized in this manner, and since the species diverged these patterns of gene expression have been maintained. Significance statementThe role of bundle sheath cells in C4 species have been studied intensively but this is not the case in leaves that use the ancestral C3 pathway. Here, we show that gene expression in the bundle sheath of rice is specialized to allow sulphate and nitrate reduction, water transport and jasmonate synthesis, and comparative analysis with Arabidopsis indicates ancient roles for bundle sheath cells in water transport, sulphur and jasmonate synthesis.

plant biology↗

bioRxiv2020

Targeted knockdown of ribulose-1, 5-bisphosphate carboxylase-oxygenase in rice mesophyll cells impact on photosynthesis and growth

We generated antisense constructs targeting two of the five Rubisco small subunit genes (OsRBCS2 and 4) which account for between 30-40% of the RBCS transcript abundance in leaf blades. The constructs were driven by a maize phosphoenolpyruvate carboxylase (PEPC) promoter known to have enriched expression in mesophyll cells (MCs). In the resulting lines leaf Rubisco protein content was reduced by between 30-50% and CO2 assimilation rate was limited under photorespiratory and non-photorespiratory conditions. A relationship between Rubisco protein content and CO2 assimilation rate was found. This was associated with a significant reduction in dry biomass accumulation and grain yield of between 37 to 70%. In addition to serving as a resource for reducing Rubisco accumulation in a cell-preferential manner, these lines allow us to characterize gene function and isoform specific suppression on photosynthesis and growth. Our results suggest that the knockdown of multiple genes is required to completely reduce Rubisco accumulation in MCs.

plant biology↗

bioRxiv2020

The rice bundle sheath produces reactive oxygen species during high light stress via NADPH oxidase

When exposed to high light plants produce reactive oxygen species (ROS). In Arabidopsis thaliana local accumulation of ROS preferentially takes place in bundle sheath strands, but little is known about how this response takes place. Using rice and the ROS probes diaminobenzidine and 2,7-dichlorodihydrofluorescein diacetate, we found that after exposure to high light, ROS were produced more rapidly in bundle sheath strands than mesophyll cells. This response was not affected either by CO2 supply or photorespiration. Consistent with these findings, deep sequencing of mRNA isolated from mesophyll or bundle sheath strands indicated balanced accumulation of transcripts encoding all major components of the photosynthetic apparatus. However, transcripts encoding several isoforms of the superoxide/H2O2-producing enzyme NADPH oxidase were more abundant in bundle sheath strands than mesophyll cells. ROS production in bundle sheath strands was reduced by blocking NADPH oxidase activity pharmacologically, but increased when the bundle sheath preferential RBOHA isoform of NADPH oxidase was over-expressed. NADPH oxidase mediated accumulation of ROS in the rice bundle sheath was detected in etiolated leaves lacking chlorophyll indicating that high light and NADPH oxidase-dependent ROS production is not dependent on photosynthesis.

plant biology↗

bioRxiv2020

Upregulation and cell specificity of C4 genes are derived from ancestral C3 gene regulatory networks

C4 photosynthesis has evolved independently in over sixty lineages and in so doing repurposed existing enzymes to drive a carbon pump that limits the RuBisCO oxygenation reaction. In all cases, gene expression is modified such that C4 proteins accumulate to levels matching those of the photosynthetic apparatus. To better understand this rewiring of gene expression we undertook RNA- and DNaseI-SEQ on de-etiolating seedlings of C4 Gynandropsis gynandra, which is sister to C3 Arabidopsis. Changes in chloroplast ultrastructure and C4 gene expression were coordinated and rapid. C3 photosynthesis and C4 genes showed similar induction patterns, but C4 genes from G. gynandra were more strongly induced than orthologs from Arabidopsis. A gene regulatory network predicted transcription factors operating at the top of the de-etiolation network, including those responding to light, act upstream of C4 genes. Light responsive elements, especially G-, E- and GT-boxes were over-represented in accessible chromatin around C4 genes. Moreover, in vivo binding of many G-, E- and GT-boxes was detected. Overall, the data support a model in which rapid and robust C4 gene expression following light exposure is generated through modifications in cis to allow integration into high-level transcriptional networks including those underpinned by conserved light responsive elements.

plant biology↗

bioRxiv2020

A partial C4 photosynthetic biochemical pathway in rice

Introduction of a C4 photosynthetic pathway into C3 rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO2 at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously expresses four of the core C4 photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME) and pyruvate phosphate dikinase (ZmPPDK), in a cell-specific manner. This led to enhanced enzyme activity but was largely neutral in its effects on photosynthetic rate and growth. Measurements of the flux of 13CO2 through photosynthetic metabolism revealed a significant increase in the incorporation of 13C into malate, consistent with increased fixation of 13CO2 via PEP carboxylase in lines expressing the maize PEPC enzyme. We also showed 13C labelling of aspartate indicating additional 13CO2 fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. However, there were no significant differences in labelling of 3-phosphoglycerate (3PGA) or phosphoenolpyruvate (PEP) indicating limited carbon flux through C4 enzymes into the Calvin-Benson cycle. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein (OsGDCH) abundance led to a photosynthetic phenotype characteristic of the reduced OsGDCH line and higher labelling of malate, aspartate and citrate. While Kranz anatomy or other anatomical modifications have not yet been installed in these plants to enable a fully functional C4 cycle, these results demonstrate for the first-time flux through the carboxylation phase of C4 metabolism in transgenic rice containing the key metabolic steps in the C4 pathway.

plant biology↗

bioRxiv2020

A role for neutral variation in the evolution of C4 photosynthesis

Convergent trait evolution is a recurrent phenomenon in all domains of the tree of life. While some convergent traits are caused by simple sequence changes, many are associated with extensive changes to the sequence and regulation of large cohorts of genes. It is unknown how organisms traverse this expansive genotype space to assemble such complex convergent phenotypes. C4 photosynthesis is a paradigm of large-scale phenotypic convergence. Conceptual and mathematical models propose that C4 photosynthesis evolved from ancestral C3 photosynthesis through sequential adaptive changes. These adaptive changes could have been rapidly assembled if modifications to the activity and abundance of enzymes of the C4 cycle was neutral in C3 plants. This neutrality would enable populations of C3 plants to maintain genotypes with expression levels of C4 enzymes analogous to those in C4 species and thus enable rapid assembly of a functional C4 cycle from naturally occurring genotypes given shared environmental selection. Here we show that there is substantial natural variation in expression of genes encoding C4 cycle enzymes between natural accessions of the C3 plant Arabidopsis thaliana. We further show through targeted transgenic experiments in the C3 crop Oryza sativa, that high expression of the majority of C4 cycle enzymes in rice is neutral with respect to growth, development, biomass and photosynthesis. Thus, substantial variation in the abundance and activity of C4 cycle enzymes is permissible within the limits of operation of C3 photosynthesis and the emergence of component parts of this complex convergent trait can be facilitated by neutral variation.

plant biology↗

bioRxiv2019

Fluorescent reporters for functional analysis in rice leaves

Fluorescent reporters have facilitated non-invasive imaging in multiple plant species and thus allowed analysis of processes ranging from gene expression and protein localization through to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualise fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee, and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins, mTurquoise2, mClover3, mNeonGreen, mKO{kappa} and tdTomato that are robustly expressed and visible in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolour imaging of different sub-cellular compartments. Overall, we conclude that mTurquoise2, mClover3, mNeonGreen, mKO{kappa} and tdTomato are suitable for high resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice.\n\nOne sentence summaryWe report five fluorescent reporters suitable for functional analysis in rice leaves.

plant biology↗

bioRxiv2019

An optimised protocol for isolation of RNA through laser capture microdissection of leaf material

Laser Capture Microdissection is a powerful tool that allows thin slices of specific cells types to be separated from one another. However, the most commonly used protocol, which involves embedding tissue in paraffin wax, results in severely degraded RNA. Yields from low abundance cell types of leaves are particularly compromised. We reasoned that the relatively high temperature used for sample embedding, and aqueous conditions associated with sample preparation prior to microdissection contribute to RNA degradation. Here we describe an optimized procedure to limit RNA degradation that is based on the use of low melting point wax as well as modifications to sample preparation prior to dissection, and isolation of paradermal, rather than transverse sections. Using this approach high quality RNA suitable for down-stream applications such as quantitative reverse transcriptase polymerase chain reactions or RNA-sequencing is recovered from microdissected bundle sheath strands and mesophyll cells of leaf tissue.

plant biology↗

bioRxiv2018

The Coordination and Jumps along C4 Photosynthesis Evolution in the Genus Flaveria

BackgroundC4 photosynthesis is a remarkable complex trait, elucidations of the evolutionary trajectory of C4 photosynthesis from its ancestral C3 pathway can help us to better understand the generic principles of complex trait evolution and guide engineering of C3 crops for higher yields. We used the genus Flaveria that contains C3, C3-C4, C4-like and C4 species as a system to study the evolution of C4 photosynthesis.\n\nResultsWe mapped transcript abundance, protein sequence, and morphological features to the phylogenetic tree of the genus Flaveria, and calculated the evolutionary correlation of different features. Besides, we predicted the relative changes of ancestral nodes of those features to illustrate the key stages during the evolution of C4 photosynthesis. Gene expression and protein sequence showed consistent modification pattern along the phylogenetic tree. High correlation coefficients ranging from 0.46 to 0.9 among gene expression, protein sequence and morphology were observed, and the greatest modification of those different features consistently occurred at the transition between C3-C4 species and C4-like species.\n\nConclusionsOur data shows highly coordinated changes in gene expression, protein sequence and morphological features. Besides, our results support an obviously evolutionary jump during the evolution of C4 metabolism.

evolutionary biology↗

bioRxiv2018

A single cis-element that controls cell-type specific expression in Arabidopsis

C4 photosynthesis evolved repeatedly from the ancestral C3 state, improving photosynthetic efficiency by ∼50%. In most C4 lineages photosynthesis is compartmented between mesophyll and bundle sheath cells but how gene expression is restricted to these cell types is poorly understood. Using the C3 model Arabidopsis thaliana we identified cis-elements and transcription factors driving expression in bundle sheath strands. Upstream of the bundle sheath preferentially expressed MYB76 gene we identified a region necessary and sufficient for expression containing two cis-elements associated with the MYC and MYB families of transcription factors. MYB76 expression is reduced in mutant alleles for each. Moreover, down-regulated genes shared by both mutants are preferentially expressed in the bundle sheath. Our findings are broadly relevant for understanding the spatial patterning of gene expression, provide specific insights into mechanisms associated with evolution of C4 photosynthesis and identify a short tuneable sequence for manipulating gene expression in the bundle sheath.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2018

Spatial photosynthesis modelling sets guidelines to constructing a viable single-cell cytoplasm-to-stroma C4 cycle

It has been proposed that introducing C4 photosynthesis into C3 crops would increase yield. The simplest scheme in- volves concentrating carbon originating from the cytosol in the chloroplast stroma of mesophyll cells without altering leaf or cell anatomy. Photosynthetic efficiency would then strongly depend on the chloroplast envelope permeability to CO2. We examine the performance of this C4 cycle with a spatial model of carbon assimilation in C3 mesophyll cell geometry, conducting a thorough exploration of parameter space relevant to C4 photosynthesis. For envelope perme- abilities below 300 {micro}m/s C4 photosynthesis has a higher quantum efficiency than C3. However, even when envelope permeability is above this threshold, the C4 pathway can provide a substantial boost to carbon assimilation with only a moderate decrease in efficiency. Depending on the available light-harvesting capacity of plastids, C4 photosynthesis could boost carbon assimilation anywhere from 20% to 100%. Gains are even more prominent under CO2 deprivation, and can be achieved in conjunction with lower investment in plastids if chloroplast surface coverage is also altered. A C4 pathway operating within individual mesophyll cells of C3 plants could hence lead to higher growth rates and better drought resistance in dry, high-sunlight climates.

plant biology↗

bioRxiv2018

Synergistic binding of bHLH transcription factors to the promoter of the maize NADP-ME gene used in C4 photosynthesis is based on an ancient code found in the ancestral C3 state

C4 photosynthesis has evolved repeatedly from the ancestral C3 state to generate a carbon concentrating mechanism that increases photosynthetic efficiency. This specialised form of photosynthesis is particularly common in the PACMAD clade of grasses, and is used by many of the worlds most productive crops. The C4 cycle is accomplished through cell-type specific accumulation of enzymes but cis-elements and transcription factors controlling C4 photosynthesis remain largely unknown. Using the NADP-Malic Enzyme (NADP-ME) gene as a model we aimed to better understand molecular mechanisms associated with the evolution of C4 photosynthesis. Two basic Helix-Loop-Helix (bHLH) transcription factors, ZmbHLH128 and ZmbHLH129, were shown to bind the C4 NADP-ME promoter from maize. These proteins form heterodimers and ZmbHLH129 impairs trans-activation by ZmbHLH128. Electrophoretic mobility shift assays indicate that a pair of cis-elements separated by a seven base pair spacer synergistically bind either ZmbHLH128 or ZmbHLH129. This pair of cis-elements is found in both C3 and C4 species of the PACMAD clade. Our analysis is consistent with this cis-element pair originating from a single motif present in the ancestral C3 state. We conclude that C4 photosynthesis has co-opted an ancient C3 regulatory code built on G-box recognition by bHLH to regulate the NADP-ME gene. More broadly, our findings also contribute to the understanding of gene regulatory networks controlling C4 photosynthesis.

plant biology↗

bioRxiv2018

Expression of SULTR2;2 in the Arabidopsis bundle sheath is mediated by a highly conserved positive regulator

The bundle sheath provides a conduit linking veins and mesophyll cells. In C3 Arabidopsis thaliana it also plays important roles in oxidative stress and sulphur metabolism. However, the mechanisms responsible for the patterns of gene expression that underpin these metabolic specialisations are poorly understood. Here we used the A. thaliana SULTR2;2 gene as a model to better understand mechanisms that restrict expression to the bundle sheath. Deletion analysis indicated that the SULTR2;2 promoter contains a short region necessary for expression in the bundle sheath. This sequence acts as a positive regulator and is tolerant to multiple consecutive deletions indicating considerable redundancy in the cis-elements involved. It is highly conserved in SULTR2;2 genes of the Brassicaceae and is functional in the distantly related C4 species Flaveria bidentis that belongs to the Asteraceae. We conclude that expression of SULTR2;2 in the bundle sheath is underpinned by a highly redundant sequence that likely represents an ancient and conserved mechanism found in families as diverse as the Asteraceae and Brassicaceae.

plant biology↗

bioRxiv2017

Droplet-based microfluidic analysis and screening of single plant cells

Droplet-based microfluidics has been used to facilitate high throughput analysis of individual prokaryote and mammalian cells. However, there is a scarcity of similar workflows applicable to rapid phenotyping of plant systems. We report on-chip encapsulation and analysis of protoplasts isolated from the emergent plant model Marchantia polymorpha at processing rates of >100,000 protoplasts per hour. We use our microfluidic system to quantify the stochastic properties of a heat-inducible promoter across a population of transgenic protoplasts to demonstrate that it has the potential to assess gene expression activity in response to environmental conditions. We further demonstrate on-chip sorting of droplets containing YFP-expressing protoplasts from wild type cells using dielectrophoresis force. This work opens the door to droplet-based microfluidic analysis of plant cells for applications ranging from high-throughput characterisation of DNA parts to single-cell genomics.

synthetic biology↗