Biology subjects

Hibberd, J. M.

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

9 recordsLinked to original sources

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↗

bioRxiv2017

A transcription factor binding atlas for photosynthesis in cereals identifies a key role for coding sequence in the regulation of gene expression

The majority of plants use C3 photosynthesis, but over sixty independent lineages of angiosperms have evolved the C4 pathway. In most C4 species, photosynthesis gene expression is compartmented between mesophyll and bundle sheath cells. We performed DNaseI-SEQ to identify genome-wide profiles of transcription factor binding in leaves of the C4 grasses Zea mays, Sorghum bicolor and Setaria italica as well as C3 Brachypodium distachyon. In C4 species, while bundle sheath strands and whole leaves shared similarity in the broad regions of DNA accessible to transcription factors, the short sequences bound varied. Transcription factor binding was prevalent in gene bodies as well as promoters, and many of these sites could represent duons that impact gene regulation in addition to amino acid sequence. Although globally there was little correlation between any individual DNaseI footprint and cell-specific gene expression, within individual species transcription factor binding to the same motifs in multiple genes provided evidence for shared mechanisms governing C4 photosynthesis gene expression. Furthermore, interspecific comparisons identified a small number of highly conserved transcription factor binding sites associated with leaves from species that diverged around 60 million years ago. These data therefore provide insight into the architecture associated with C4 photosynthesis gene expression in particular and characteristics of transcription factor binding in cereal crops in general.\n\nOne sentence summaryGenome-wide patterns of transcription factor binding in vivo defined by DNaseI for leaves of C3 and C4 grasses

plant biology↗

bioRxiv2017

Ancestral Reconstruction and C3 Bundle Sheath Transcript Abundance in the Paniceae Grasses Indicate the Foundations for all Three Biochemical C4 Sub-Types Were Likely Present in the Most Recent Ancestor

In C4 plants, the enzymatic machinery underpinning photosynthesis can vary, with, for example, three distinct C4 acid decarboxylases being used to release CO2 in the vicinity of RuBisCO. For decades, these decarboxylases have been used to classify C4 species into three biochemical sub-types. However, more recently the notion that C4 species mix and match C4 acid decarboxylases has increased in popularity and, as a consequence, the validity of specific biochemical sub-types has been questioned. Using five species from the grass tribe Paniceae, we show that, while in some species transcripts encoding multiple C4 acid decarboxylases accumulate, in others, transcript abundance and enzyme activity is almost entirely from one decarboxylase. In addition, the development of a bundle sheath isolation procedure for a close C3 species in the Paniceae enables the preliminary exploration of C4 sub-type evolution.

plant biology↗

bioRxiv2016

Ancient coding sequences underpin the spatial patterning of gene expression in C4 leaves

Photosynthesis is compromised in most plants because an enzymatic side-reaction fixes O2 instead of CO2. The energetic cost of oxygenation led to the evolution of C4 photosynthesis. In almost all C4 leaves compartmentation of photosynthesis between cells reduces oxygenation and so increases photosynthetic efficiency. Here we report that spatial expression of most C4 genes is controlled by intragenic cis-elements rather than promoter sequence. Two DNA motifs that cooperatively specify the patterning of genes required for C4 photosynthesis are identified. They are conserved in plants and algae that use the ancestral C3 pathway. As these motifs are located in exons they represent duons determining both gene expression and amino acid sequence. Our findings provide functional evidence for the importance of transcription factors recognising coding sequence as previously defined by genome-wide binding studies. Furthermore, they indicate that C4 evolution is based on ancient DNA motifs found in exonic sequence.

plant biology↗

bioRxiv2016

Shared characteristics underpinning C4 leaf maturation derived from analysis of multiple C3 and C4 species of Flaveria

Most terrestrial plants use C3 photosynthesis to fix carbon. In multiple plant lineages a modified system known as C4 photosynthesis has evolved. To better understand the molecular patterns associated with induction of C4 photosynthesis the genus Flaveria that contains C3 and C4 species was used. A base to tip maturation gradient of leaf anatomy was defined, and RNA sequencing was undertaken along this gradient for two C3 and two C4 Flaveria species. Key C4 traits including vein density, mesophyll and bundle sheath cross-sectional area, chloroplast ultrastructure, and abundance of transcripts encoding proteins of C4 photosynthesis were quantified. Candidate genes underlying each of these C4 characteristics were identified. Principal Components Analysis indicated that leaf maturation and then photosynthetic pathway were responsible for the greatest amount of variation in transcript abundance. Photosynthesis genes were over-represented for a prolonged period in the C4 species. Through comparison with publically available datasets we identify a small number of transcriptional regulators that have been up-regulated in diverse C4 species. The analysis identifies similar patterns of expression in independent C4 lineages and so indicates that the complex C4 pathway is associated with parallel as well as convergent evolution.\n\nHighlightWe identify transcription factors that show conserved patterns of expression in multiple 29 C4 species, both within the Flaveria genus, but also in more distantly related C4 plants.

plant biology↗