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LSSS 2017-2018


Life Sciences Seminar Series


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Jane Langdale

University of Oxford

The C4 rice project – an agricultural grand challenge

Selected Publications

Genetic Regulation of the 2D to 3D Growth Transition in the Moss Physcomitrella patens.Moody LA, Kelly S, Rabbinowitsch E, Langdale JA
Curr Biol 2018 Feb 5; 28(3):473-478.e5


One of the most important events in the history of life on earth was the colonization of land by plants; this transition coincided with and was most likely enabled by the evolution of 3-dimensional (3D) growth. Today, the diverse morphologies exhibited across the terrestrial biosphere arise from the differential regulation of 3D growth processes during development. In many plants, 3D growth is initiated during the first few divisions of the zygote, and therefore, the genetic basis cannot be dissected because mutants do not survive. However, in mosses, which are representatives of the earliest land plants, 3D shoot growth is preceded by a 2D filamentous phase that can be maintained indefinitely. Here, we used the moss Physcomitrella patens to identify genetic regulators of the 2D to 3D transition. Mutant screens yielded individuals that could only grow in 2D, and through an innovative strategy that combined somatic hybridization with bulk segregant analysis and genome sequencing, the causative mutation was identified in one of them. The NO GAMETOPHORES 1 (NOG1) gene, which encodes a ubiquitin-associated protein, is present only in land plant genomes. In mutants that lack PpNOG1 function, transcripts encoding 3D-promoting PpAPB transcription factors [1] are significantly reduced, and apical initial cells specified for 3D growth are not formed. PpNOG1 acts at the earliest identified stage of the 2D to 3D transition, possibly through degradation of proteins that suppress 3D growth. The acquisition of NOG1 function in land plants could thus have enabled the evolution and development of 3D morphology.

SHORTROOT-Mediated Increase in Stomatal Density Has No Impact on Photosynthetic Efficiency.Schuler ML, Sedelnikova OV, Walker BJ, Westhoff P, Langdale JA
Plant Physiol 2018 Jan; 176(1):757-772


The coordinated positioning of veins, mesophyll cells, and stomata across a leaf is crucial for efficient gas exchange and transpiration and, therefore, for overall function. In monocot leaves, stomatal cell files are positioned at the flanks of underlying longitudinal leaf veins, rather than directly above or below. This pattern suggests either that stomatal formation is inhibited in epidermal cells directly in contact with the vein or that specification is induced in cell files beyond the vein. The SHORTROOT pathway specifies distinct cell types around the vasculature in subepidermal layers of both root and shoots, with cell type identity determined by distance from the vein. To test whether the pathway has the potential to similarly pattern epidermal cell types, we expanded the expression domain of the rice (ssp)gene, which we show is restricted to developing leaf veins, to include bundle sheath cells encircling the vein. In transgenic lines, which were generated using the orthologousgene to avoid potential silencing of, stomatal cell files were observed both in the normal position and in more distant positions from the vein. Contrary to theoretical predictions, and to phenotypes observed in eudicot leaves, the increase in stomatal density did not enhance photosynthetic capacity or increase mesophyll cell density. Collectively, these results suggest that the SHORTROOT pathway may coordinate the positioning of veins and stomata in monocot leaves and that distinct mechanisms may operate in monocot and eudicot leaves to coordinate stomatal patterning with the development of underlying mesophyll cells.

Re-creation of a Key Step in the Evolutionary Switch from C3 to C4 Leaf Anatomy.Wang P, Khoshravesh R, Karki S, Tapia R, Balahadia CP, Bandyopadhyay A, Quick WP, Furbank R, Sage TL, Langdale JA
Curr Biol 2017 Nov 6; 27(21):3278-3287.e6


The Cphotosynthetic pathway accounts for ∼25% of primary productivity on the planet despite being used by only 3% of species. Because Cplants are higher yielding than Cplants, efforts are underway to introduce the Cpathway into the Ccrop rice. This is an ambitious endeavor; however, the Cpathway evolved from Con multiple independent occasions over the last 30 million years, and steps along the trajectory are evident in extant species. One approach toward engineering Crice is to recapitulate this trajectory, one of the first steps of which was a change in leaf anatomy. The transition from Cto so-called "proto-Kranz" anatomy requires an increase in organelle volume in sheath cells surrounding leaf veins. Here we induced chloroplast and mitochondrial development in rice vascular sheath cells through constitutive expression of maize GOLDEN2-LIKE genes. Increased organelle volume was accompanied by the accumulation of photosynthetic enzymes and by increased intercellular connections. This suite of traits reflects that seen in "proto-Kranz" species, and, as such, a key step toward engineering Crice has been achieved.

Candidate regulators of Early Leaf Development in Maize Perturb Hormone Signalling and Secondary Cell Wall Formation When Constitutively Expressed in Rice.Wang P, Karki S, Biswal AK, Lin HC, Dionora MJ, Rizal G, Yin X, Schuler ML, Hughes T, Fouracre JP, Jamous BA, Sedelnikova O, Lo SF, Bandyopadhyay A, Yu SM, Kelly S, Quick WP, Langdale JA
Sci Rep 2017 Jul 3; 7(1):4535


All grass leaves are strap-shaped with a series of parallel veins running from base to tip, but the distance between each pair of veins, and the cell-types that develop between them, differs depending on whether the plant performs Cor Cphotosynthesis. As part of a multinational effort to introduce Ctraits into rice to boost crop yield, candidate regulators of Cleaf anatomy were previously identified through an analysis of maize leaf transcriptomes. Here we tested the potential of 60 of those candidate genes to alter leaf anatomy in rice. In each case, transgenic rice lines were generated in which the maize gene was constitutively expressed. Lines grouped into three phenotypic classes: (1) indistinguishable from wild-type; (2) aberrant shoot and/or root growth indicating possible perturbations to hormone homeostasis; and (3) altered secondary cell wall formation. One of the genes in class 3 defines a novel monocot-specific family. None of the genes were individually sufficient to induce C-like vein patterning or cell-type differentiation in rice. A better understanding of gene function in Cplants is now needed to inform more sophisticated engineering attempts to alter leaf anatomy in Cplants.

Nonreciprocal complementation of KNOX gene function in land plants.Frangedakis E, Saint-Marcoux D, Moody LA, Rabbinowitsch E, Langdale JA
New Phytol 2017 Oct; 216(2):591-604


Class I KNOTTED-LIKE HOMEOBOX (KNOX) proteins regulate development of the multicellular diploid sporophyte in both mosses and flowering plants; however, the morphological context in which they function differs. In order to determine how Class I KNOX function was modified as land plants evolved, phylogenetic analyses and cross-species complementation assays were performed. Our data reveal that a duplication within the charophyte sister group to land plants led to distinct Class I and Class II KNOX gene families. Subsequently, Class I sequences diverged substantially in the nonvascular bryophyte groups (liverworts, mosses and hornworts), with moss sequences being most similar to those in vascular plants. Despite this similarity, moss mutants were not complemented by vascular plant KNOX genes. Conversely, the Arabidopsis brevipedicellus (bp-9) mutant was complemented by the PpMKN2 gene from the moss Physcomitrella patens. Lycophyte KNOX genes also complemented bp-9 whereas fern genes only partially complemented the mutant. This lycophyte/fern distinction is mirrored in the phylogeny of KNOX-interacting BELL proteins, in that a gene duplication occurred after divergence of the two groups. Together, our results imply that the moss MKN2 protein can function in a broader developmental context than vascular plant KNOX proteins, the narrower scope having evolved progressively as lycophytes, ferns and flowering plants diverged.

Finding the genes to build C4 rice.Wang P, Vlad D, Langdale JA
Curr Opin Plant Biol 2016 Jun; 31:44-50


Rice, a C3 crop, is a staple food for more than half of the world's population, with most consumers living in developing countries. Engineering C4 photosynthetic traits into rice is increasingly suggested as a way to meet the 50% yield increase that is predicted to be needed by 2050. Advances in genome-wide deep-sequencing, gene discovery and genome editing platforms have brought the possibility of engineering a C3 to C4 conversion closer than ever before. Because C4 plants have evolved independently multiple times from C3 origins, it is probably that key genes and gene regulatory networks that regulate C4 were recruited from C3 ancestors. In the past five years there have been over 20 comparative transcriptomic studies published that aimed to identify these recruited C4 genes and regulatory mechanisms. Here we present an overview of what we have learned so far and preview the efforts still needed to provide a practical blueprint for building C4 rice.

Ferns: the missing link in shoot evolution and development.Plackett AR, Di Stilio VS, Langdale JA
Front Plant Sci 2015; 6:972


Shoot development in land plants is a remarkably complex process that gives rise to an extreme diversity of forms. Our current understanding of shoot developmental mechanisms comes almost entirely from studies of angiosperms (flowering plants), the most recently diverged plant lineage. Shoot development in angiosperms is based around a layered multicellular apical meristem that produces lateral organs and/or secondary meristems from populations of founder cells at its periphery. In contrast, non-seed plant shoots develop from either single apical initials or from a small population of morphologically distinct apical cells. Although developmental and molecular information is becoming available for non-flowering plants, such as the model moss Physcomitrella patens, making valid comparisons between highly divergent lineages is extremely challenging. As sister group to the seed plants, the monilophytes (ferns and relatives) represent an excellent phylogenetic midpoint of comparison for unlocking the evolution of shoot developmental mechanisms, and recent technical advances have finally made transgenic analysis possible in the emerging model fern Ceratopteris richardii. This review compares and contrasts our current understanding of shoot development in different land plant lineages with the aim of highlighting the potential role that the fern C. richardii could play in shedding light on the evolution of underlying genetic regulatory mechanisms.

Two forward genetic screens for vein density mutants in sorghum converge on a cytochrome P450 gene in the brassinosteroid pathway.Rizal G, Thakur V, Dionora J, Karki S, Wanchana S, Acebron K, Larazo N, Garcia R, Mabilangan A, Montecillo F, Danila F, Mogul R, Pablico P, Leung H, Langdale JA, Sheehy J, Kelly S, Quick WP
Plant J 2015 Oct; 84(2):257-66


The specification of vascular patterning in plants has interested plant biologists for many years. In the last decade a new context has emerged for this interest. Specifically, recent proposals to engineer C(4) traits into C(3) plants such as rice require an understanding of how the distinctive venation pattern in the leaves of C(4) plants is determined. High vein density with Kranz anatomy, whereby photosynthetic cells are arranged in encircling layers around vascular bundles, is one of the major traits that differentiate C(4) species from C(3) species. To identify genetic factors that specify C(4) leaf anatomy, we generated ethyl methanesulfonate- and γ-ray-mutagenized populations of the C(4) species sorghum (Sorghum bicolor), and screened for lines with reduced vein density. Two mutations were identified that conferred low vein density. Both mutations segregated in backcrossed F(2) populations as homozygous recessive alleles. Bulk segregant analysis using next-generation sequencing revealed that, in both cases, the mutant phenotype was associated with mutations in the CYP90D2 gene, which encodes an enzyme in the brassinosteroid biosynthesis pathway. Lack of complementation in allelism tests confirmed this result. These data indicate that the brassinosteroid pathway promotes high vein density in the sorghum leaf, and suggest that differences between C(4) and C(3) leaf anatomy may arise in part through differential activity of this pathway in the two leaf types.

Establishment of Anthoceros agrestis as a model species for studying the biology of hornworts.Szövényi P, Frangedakis E, Ricca M, Quandt D, Wicke S, Langdale JA
BMC Plant Biol 2015 Apr 9; 15:98


Plants colonized terrestrial environments approximately 480 million years ago and have contributed significantly to the diversification of life on Earth. Phylogenetic analyses position a subset of charophyte algae as the sister group to land plants, and distinguish two land plant groups that diverged around 450 million years ago - the bryophytes and the vascular plants. Relationships between liverworts, mosses hornworts and vascular plants have proven difficult to resolve, and as such it is not clear which bryophyte lineage is the sister group to all other land plants and which is the sister to vascular plants. The lack of comparative molecular studies in representatives of all three lineages exacerbates this uncertainty. Such comparisons can be made between mosses and liverworts because representative model organisms are well established in these two bryophyte lineages. To date, however, a model hornwort species has not been available.

Identification of reference genes for real-time quantitative PCR experiments in the liverwort Marchantia polymorpha.Saint-Marcoux D, Proust H, Dolan L, Langdale JA
PLoS One 2015; 10(3):e0118678


Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. MpAPT and MpACT showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that MpAPT and MpACT are suitable reference genes in M. polymorpha and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data.

Laser capture microdissection in Ectocarpus siliculosus: the pathway to cell-specific transcriptomics in brown algae.Saint-Marcoux D, Billoud B, Langdale JA, Charrier B
Front Plant Sci 2015; 6:54


Laser capture microdissection (LCM) facilitates the isolation of individual cells from tissue sections, and when combined with RNA amplification techniques, it is an extremely powerful tool for examining genome-wide expression profiles in specific cell-types. LCM has been widely used to address various biological questions in both animal and plant systems, however, no attempt has been made so far to transfer LCM technology to macroalgae. Macroalgae are a collection of widespread eukaryotes living in fresh and marine water. In line with the collective effort to promote molecular investigations of macroalgal biology, here we demonstrate the feasibility of using LCM and cell-specific transcriptomics to study development of the brown alga Ectocarpus siliculosus. We describe a workflow comprising cultivation and fixation of algae on glass slides, laser microdissection, and RNA amplification. To illustrate the effectiveness of the procedure, we show qPCR data and metrics obtained from cell-specific transcriptomes generated from both upright and prostrate filaments of Ectocarpus.