Biology subjects

Reyna-Llorens, I.

Publications and source records attributed to Reyna-Llorens, I..

9 recordsLinked to original sources

bioRxiv2026

The demonstration of a single origin for nodule evolution, with nodule engineering in a non-nodulating species

The nitrogen-fixing root-nodule symbiosis provides a sustainable source of nitrogen for plants within the Nitrogen (N)-fixing clade (NFC). A debate has raged over whether nodulation evolved once, with many losses or multiple times following a predisposition event. Here we demonstrate that nodule-organogenesis is fully conserved between an actinorhizal nodulator Datisca glomerata and the legume Medicago truncatula, showing entirely conserved programmes for Nodule INception (NIN)-controlled development leading to nodule emergence. Convergent losses of N-fixation within the NFC is associated with loss of NIN and we show the engineering of nodule-like development into strawberry, a non-nodulating member of the NFC, through the repair of NIN-functionality. Similar NIN-engineering resulted in altered-root developmental responses in barley. Our work is consistent with the single-gain hypothesis, where repair of NIN can recapitulate nodules in species within the NFC, demonstrating that understanding the ancestral state of nodulation facilitates its engineering.

plant biology↗

bioRxiv2023

Compartmentation of photosynthesis gene expression between mesophyll and bundle sheath cells of C4 maize is dependent on time of day

Compared with the ancestral C3 state, C4 photosynthesis enables higher rates of photosynthesis as well as improved water and nitrogen use efficiencies. In both C3 and C4 plants rates of photosynthesis increase with light intensity and so are maximal around midday. We report that in the absence of light or temperature fluctuations, photosynthesis in maize peaks in the middle of the subjective photoperiod. To investigate molecular processes associated with these changes, we undertook RNA-sequencing of maize mesophyll and bundle sheath strands over a 24-hour time-course. Cell-preferential expression of C4 cycle genes was strongest between six and ten hours after dawn when rates of photosynthesis were highest. For the bundle sheath, DNA motif enrichment and gene co-expression analyses suggested members of the DOF and MADS-domain transcription factor families mediate diurnal fluctuations in C4 gene expression, and trans-activation assays in planta confirmed their ability to activate promoter fragments from bundle sheath expressed genes. The work thus identifies transcriptional regulators as well as peaks in cell-specific C4 gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.

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

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↗

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↗

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↗

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↗