The WIP6 transcription factor TOO MANY LATERALS specifies vein type in C4 and C3 grass leaves.

Grass leaves are invariantly strap shaped with an elongated distal blade and a proximal sheath that wraps around the stem. Underpinning this shape is a scaffold of leaf veins, most of which extend in parallel along the proximo-distal leaf axis. Differences between species are apparent both in the vein types that develop and in the distance between veins across the medio-lateral leaf axis. A prominent engineering goal is to increase vein density in leaves of C3 photosynthesizing species to facilitate the introduction of the more efficient C4 pathway. Here, we discover that the WIP6 transcription factor TOO MANY LATERALS (TML) specifies vein rank in both maize (C4) and rice (C3). Loss-of-function tml mutations cause large lateral veins to develop in positions normally occupied by smaller intermediate veins, and TML transcript localization in wild-type leaves is consistent with a role in suppressing lateral vein development in procambial cells that form intermediate veins. Attempts to manipulate TML function in rice were unsuccessful because transgene expression was silenced, suggesting that precise TML expression is essential for shoot viability. This finding may reflect the need to prevent the inappropriate activation of downstream targets or, given that transcriptome analysis revealed altered cytokinin and auxin signaling profiles in maize tml mutants, the need to prevent local or general hormonal imbalances. Importantly, rice tml mutants display an increased occupancy of veins in the leaf, providing a step toward an anatomical chassis for C4 engineering. Collectively, a conserved mechanism of vein rank specification in grass leaves has been revealed.

The bHLH transcription factor OsPRI1 activates the Setaria viridis PEPC1 promoter in rice.

Photosynthetic efficiency is reduced by the dual role of Rubisco, which acts either as a carboxylase or as an oxygenase, the latter leading to photorespiration. C4 photosynthesis evolved as a carbon-concentrating mechanism to reduce photorespiration. To engineer C4 into a C3 plant, it is essential to understand how C4 genes, such as phosphoenolpyruvate carboxylase (PEPC1), are regulated to be expressed at high levels and in a cell-specific manner. Yeast one-hybrid screening was used to show that OsPRI1, a rice bHLH transcription factor involved in iron homeostasis, binds to the Setaria viridis PEPC1 promoter. This promoter drives mesophyll-specific gene expression in rice. The role of OsPRI1 in planta was characterized using a rice line harbouring SvPEPC1pro ::GUS. We show that OsPRI1 activates the S. viridis PEPC1 promoter by binding to an N-box in the proximal promoter, and that GUS activity is highly reduced in SvPEPC1pro ::GUS lines when OsPRI1 is mutated. Cross-species comparisons showed that the SvPRI1 homolog binds to the SvPEPC1 promoter but the maize ZmPRI1 does not bind to the ZmPEPC1 promoter. Our results suggest that elements of the iron homeostasis pathway were co-opted to regulate PEPC1 gene expression during the evolution of some but not all C4 species.

GOLDEN2-like1 is sufficient but not necessary for chloroplast biogenesis in mesophyll cells of C4 grasses.

Chloroplasts are the site of photosynthesis. In land plants, chloroplast biogenesis is regulated by a family of transcription factors named GOLDEN2-like (GLK). In C4 grasses, it has been hypothesized that genome duplication events led to the sub-functionalization of GLK paralogs (GLK1 and GLK2) to control chloroplast biogenesis in two distinct cell types: mesophyll and bundle sheath cells. Although previous characterization of golden2 (g2) mutants in maize has demonstrated a role for GLK2 paralogs in regulating chloroplast biogenesis in bundle sheath cells, the function of GLK1 has remained elusive. Here we show that, contrary to expectations, GLK1 is not required for chloroplast biogenesis in mesophyll cells of maize. Comparisons between maize and Setaria viridis, which represent two independent C4 origins within the Poales, further show that the role of GLK paralogs in controlling chloroplast biogenesis in mesophyll and bundle sheath cells differs between species. Despite these differences, complementation analysis revealed that GLK1 and GLK2 genes from maize are both sufficient to restore functional chloroplast development in mesophyll and bundle sheath cells of S. viridis mutants. Collectively our results suggest an evolutionary trajectory in C4 grasses whereby both orthologs retained the ability to induce chloroplast biogenesis but GLK2 adopted a more prominent developmental role, particularly in relation to chloroplast activation in bundle sheath cells.

Discovery of the bicycle gene family provides new insights into insect manipulation of plant development during gall induction.

Galls are complex structures that develop from plant tissue, providing protection and food for gall-forming organisms, such as insects or mites. However, the molecules used by insects or mites to manipulate plant development have proved elusive. A landmark study has tracked down a gene in a gall-forming aphid that controls whether galls on witch hazel are green or red. The 'green allele' is strongly expressed in aphid salivary glands and represses plant genes used for red color formation. Excitingly, the gene product is part of a large suite of proteins that aphids may use to interact with plant biology.

Mutations in NAKED-ENDOSPERM IDD genes reveal functional interactions with SCARECROW during leaf patterning in C4 grasses.

Leaves comprise a number of different cell-types that are patterned in the context of either the epidermal or inner cell layers. In grass leaves, two distinct anatomies develop in the inner leaf tissues depending on whether the leaf carries out C3 or C4 photosynthesis. In both cases a series of parallel veins develops that extends from the leaf base to the tip but in ancestral C3 species veins are separated by a greater number of intervening mesophyll cells than in derived C4 species. We have previously demonstrated that the GRAS transcription factor SCARECROW (SCR) regulates the number of photosynthetic mesophyll cells that form between veins in the leaves of the C4 species maize, whereas it regulates the formation of stomata in the epidermal leaf layer in the C3 species rice. Here we show that SCR is required for inner leaf patterning in the C4 species Setaria viridis but in this species the presumed ancestral stomatal patterning role is also retained. Through a comparative mutant analysis between maize, setaria and rice we further demonstrate that loss of NAKED-ENDOSPERM (NKD) INDETERMINATE DOMAIN (IDD) protein function exacerbates loss of function scr phenotypes in the inner leaf tissues of maize and setaria but not rice. Specifically, in both setaria and maize, scr;nkd mutants exhibit an increased proportion of fused veins with no intervening mesophyll cells. Thus, combined action of SCR and NKD may control how many mesophyll cells are specified between veins in the leaves of C4 but not C3 grasses. Together our results provide insight into the evolution of cell patterning in grass leaves and demonstrate a novel patterning role for IDD genes in C4 leaves.

Climate change challenges, plant science solutions.

Climate change is a defining challenge of the 21st century, and this decade is a critical time for action to mitigate the worst effects on human populations and ecosystems. Plant science can play an important role in developing crops with enhanced resilience to harsh conditions (e.g. heat, drought, salt stress, flooding, disease outbreaks) and engineering efficient carbon-capturing and carbon-sequestering plants. Here, we present examples of research being conducted in these areas and discuss challenges and open questions as a call to action for the plant science community.

DEFECTIVELY ORGANIZED TRIBUTARIES 5 is not required for leaf venation patterning in Arabidopsis thaliana.

The search for genetic regulators of leaf venation patterning started over 30 years ago, primarily focused on mutant screens in the eudicotyledon Arabidopsis thaliana. Developmental perturbations in either cotyledons or true leaves led to the identification of transcription factors required to elaborate the characteristic reticulated vein network. An ortholog of one of these, the C2H2 zinc finger protein DEFECTIVELY ORGANIZED TRIBUTARIES 5 (AtDOT5), was recently identified through transcriptomics as a candidate regulator of parallel venation in maize (Zea mays) leaves. To elucidate how AtDOT5 regulates vein patterning, we generated three independent loss-of-function mutations by gene editing in Arabidopsis. Surprisingly, none of them exhibited any obvious phenotypic perturbations. To reconcile our findings with earlier reports, we re-evaluated the original Atdot5-1 and Atdot5-2 alleles. By genome sequencing, we show that reported mutations at the Atdot5-1 locus are actually polymorphisms between Landsberg erecta and Columbia ecotypes, and that other mutations present in the background most likely cause the pleiotropic mutant phenotype observed. We further show that a T-DNA insertion in the Atdot5-2 locus has no impact on leaf venation patterns when segregated from other T-DNA insertions present in the original line. We thus conclude that AtDOT5 plays no role in leaf venation patterning in Arabidopsis.

A single promoter-TALE system for tissue-specific and tuneable expression of multiple genes in rice.

In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems-dTALE1-STAP1 and dTALE2-STAP2-can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.

SCARECROW is deployed in distinct contexts during rice and maize leaf development.

The flexible deployment of developmental regulators is an increasingly appreciated aspect of plant development and evolution. The GRAS transcription factor SCARECROW (SCR) regulates the development of the endodermis in Arabidopsis and maize roots, but during leaf development it regulates the development of distinct cell types; bundle-sheath in Arabidopsis and mesophyll in maize. In rice, SCR is implicated in stomatal patterning, but it is unknown whether this function is additional to a role in inner leaf patterning. Here, we demonstrate that two duplicated SCR genes function redundantly in rice. Contrary to previous reports, we show that these genes are necessary for stomatal development, with stomata virtually absent from leaves that are initiated after germination of mutants. The stomatal regulator OsMUTE is downregulated in Osscr1;Osscr2 mutants, indicating that OsSCR acts early in stomatal development. Notably, Osscr1;Osscr2 mutants do not exhibit the inner leaf patterning perturbations seen in Zmscr1;Zmscr1h mutants, and Zmscr1;Zmscr1h mutants do not exhibit major perturbations in stomatal patterning. Taken together, these results indicate that SCR was deployed in different developmental contexts after the divergence of rice and maize around 50 million years ago.

Flip-Flap: A Simple Dual-View Imaging Method for 3D Reconstruction of Thick Plant Samples.

Plant development is a complex process that relies on molecular and cellular events being co-ordinated in space and time. Microscopy is one of the most powerful tools available to investigate this spatiotemporal complexity. One step towards a better understanding of complexity in plants would be the acquisition of 3D images of entire organs. However, 3D imaging of intact plant samples is not always simple and often requires expensive and/or non-trivial approaches. In particular, the inner tissues of thick samples are challenging to image. Here, we present the Flip-Flap method, a simple imaging protocol to produce 3D images of cleared plant samples at the organ scale. This method allows full 3D reconstruction of plant organs suitable for 3D segmentation and further related analysis and can be easily handled by relatively inexperienced microscopists.

Developmental regulation of leaf venation patterns: monocot versus eudicots and the role of auxin.

Organisation and patterning of the vascular network in land plants varies in different taxonomic, developmental and environmental contexts. In leaves, the degree of vascular strand connectivity influences both light and CO2 harvesting capabilities as well as hydraulic capacity. As such, developmental mechanisms that regulate leaf venation patterning have a direct impact on physiological performance. Development of the leaf venation network requires the specification of procambial cells within the ground meristem of the primordium and subsequent proliferation and differentiation of the procambial lineage to form vascular strands. An understanding of how diverse venation patterns are manifest therefore requires mechanistic insight into how procambium is dynamically specified in a growing leaf. A role for auxin in this process was identified many years ago, but questions remain. In this review we first provide an overview of the diverse venation patterns that exist in land plants, providing an evolutionary perspective. We then focus on the developmental regulation of leaf venation patterns in angiosperms, comparing patterning in eudicots and monocots, and the role of auxin in each case. Although common themes emerge, we conclude that the developmental mechanisms elucidated in eudicots are unlikely to fully explain how parallel venation patterns in monocot leaves are elaborated.

Conditional stomatal closure in a fern shares molecular features with flowering plant active stomatal responses.

Stomata evolved as plants transitioned from water to land, enabling carbon dioxide uptake and water loss to be controlled. In flowering plants, the most recently divergent land plant lineage, stomatal pores actively close in response to drought. In this response, the phytohormone abscisic acid (ABA) triggers signaling cascades that lead to ion and water loss in the guard cells of the stomatal complex, causing a reduction in turgor and pore closure. Whether this stimulus-response coupling pathway acts in other major land plant lineages is unclear, with some investigations reporting that stomatal closure involves ABA but others concluding that closure is passive. Here, we show that in the model fern Ceratopteris richardii active stomatal closure is conditional on sensitization by pre-exposure to either low humidity or exogenous ABA and is promoted by ABA. RNA-seq analysis and de novo transcriptome assembly reconstructed the protein-coding complement of the C. richardii genome, with coverage comparable to other plant models, enabling transcriptional signatures of stomatal sensitization and closure to be inferred. In both cases, changes in abundance of homologs of ABA, Ca2+, and ROS-related signaling components were observed, suggesting that the closure-response pathway is conserved in ferns and flowering plants. These signatures further suggested that sensitization is achieved by lowering the threshold required for a subsequent closure-inducing signal to trigger a response. We conclude that the canonical signaling network for active stomatal closure functioned in at least a rudimentary form in the stomata of the last common ancestor of ferns and flowering plants.

NO GAMETOPHORES 2 Is a Novel Regulator of the 2D to 3D Growth Transition in the Moss Physcomitrella patens.

The colonization of land by plants was one of the most transformative events in the history of life on Earth. The transition from water, which coincided with and was likely facilitated by the evolution of three-dimensional (3D) growth, enabled the generation of morphological diversity on land. In many plants, the transition from two-dimensional (2D) to 3D growth occurs during embryo development. However, in the early divergent moss Physcomitrella patens, 3D growth is preceded by an extended filamentous phase that can be maintained indefinitely. Here, we describe the identification of the cytokinin-responsive NO GAMETOPHORES 2 (PpNOG2) gene, which encodes a shikimate o-hydroxycinnamoyltransferase. In mutants lacking PpNOG2 function, transcript levels of CLAVATA and SCARECROW genes are significantly reduced, excessive gametophore initial cells are produced, and buds undergo premature developmental arrest. Mutants also exhibit misregulation of auxin-responsive genes. Our results suggest that PpNOG2 functions in the ascorbic acid pathway leading to cuticle formation and that NOG2-related genes were co-opted into the lignin biosynthesis pathway after the divergence of bryophytes and vascular plants. We present a revised model of 3D growth in which PpNOG2 comprises part of a feedback mechanism that is required for the modulation of gametophore initial cell frequency. We also propose that the 2D to 3D growth transition in P. patens is underpinned by complex auxin-cytokinin crosstalk that is regulated, at least in part, by changes in flavonoid metabolism.

Installation of C4 photosynthetic pathway enzymes in rice using a single construct.

Introduction of a C4 photosynthetic mechanism into C3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two-cell metabolic prototype for an NADP-malic enzyme type C4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP-malate dehydrogenase, pyruvate orthophosphate dikinase and NADP-malic enzyme from Zea mays, driven by cell-preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13 CO2 labelling demonstrated a 10-fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP-malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C4 proteins introduced thus far, a functional C4 pathway is achievable in rice.

SCARECROW gene function is required for photosynthetic development in maize.

C4 photosynthesis in grasses relies on a specialized leaf anatomy. In maize, this "Kranz" leaf anatomy is patterned in part by the duplicated SCARECROW (SCR) genes ZmSCR1 and ZmSCR1h. Here we show that in addition to patterning defects, chlorophyll content and levels of transcripts encoding Golden2-like regulators of chloroplast development are significantly lower in Zmscr1; Zmscr1h mutants than in wild-type. These perturbations are not associated with changes in chloroplast number, size, or ultrastructure. However, the maximum rates of carboxylation by ribulose bisphosphate carboxylase/oxygenase (RuBisCO, V cmax) and phosphoenolpyruvate carboxylase (PEPC, V pmax) are both reduced, leading to perturbed plant growth. The CO2 compensation point and 13C‰ of Zmscr1;Zmscr1h plants are both normal, indicating that a canonical C4 cycle is operating, albeit at reduced overall capacity. Taken together, our results reveal that the maize SCR genes, either directly or indirectly, play a role in photosynthetic development. SIGNIFICANCE STATEMENT: SCARECROW (SCR) is one of the best studied plant developmental regulators, however, its role in downstream plant physiology is less well-understood. Here, we have demonstrated that SCR is required to establish and/or maintain photosynthetic capacity in maize leaves.

Single Nucleotide Polymorphism Charting of P. patens Reveals Accumulation of Somatic Mutations During in vitro Culture on the Scale of Natural Variation by Selfing.

Introduction: Physcomitrium patens (Hedw.) Mitten (previously known as Physcomitrella patens) was collected by H.L.K. Whitehouse in Gransden Wood (Huntingdonshire, United Kingdom) in 1962 and distributed across the globe starting in 1974. Hence, the Gransden accession has been cultured in vitro in laboratories for half a century. Today, there are more than 13 different pedigrees derived from the original accession. Additionally, accessions from other sites worldwide were collected during the last decades. Methods and Results: In this study, 250 high throughput RNA sequencing (RNA-seq) samples and 25 gDNA samples were used to detect single nucleotide polymorphisms (SNPs). Analyses were performed using five different P. patens accessions and 13 different Gransden pedigrees. SNPs were overlaid with metadata and known phenotypic variations. Unique SNPs defining Gransden pedigrees and accessions were identified and experimentally confirmed. They can be successfully employed for PCR-based identification. Conclusion: We show independent mutations in different Gransden laboratory pedigrees, demonstrating that somatic mutations occur and accumulate during in vitro culture. The frequency of such mutations is similar to those observed in naturally occurring populations. We present evidence that vegetative propagation leads to accumulation of deleterious mutations, and that sexual reproduction purges those. Unique SNP sets for five different P. patens accessions were isolated and can be used to determine individual accessions as well as Gransden pedigrees. Based on that, laboratory methods to easily determine P. patens accessions and Gransden pedigrees are presented.

Maize GOLDEN2-LIKE genes enhance biomass and grain yields in rice by improving photosynthesis and reducing photoinhibition.

Photosynthetic efficiency is a major target for improvement of crop yield potential under agricultural field conditions. Inefficiencies can occur in many steps of the photosynthetic process, from chloroplast biogenesis to functioning of the light harvesting and carbon fixation reactions. Nuclear-encoded GOLDEN2-LIKE (GLK) transcription factors regulate some of the earliest steps by activating target genes encoding chloroplast-localized and photosynthesis-related proteins. Here we show that constitutive expression of maize GLK genes in rice leads to enhanced levels of chlorophylls and pigment-protein antenna complexes, and that these increases lead to improved light harvesting efficiency via photosystem II in field-grown plants. Increased levels of xanthophylls further buffer the negative effects of photoinhibition under high or fluctuating light conditions by facilitating greater dissipation of excess absorbed energy as heat. Significantly, the enhanced photosynthetic capacity of field-grown transgenic plants resulted in increased carbohydrate levels and a 30-40% increase in both vegetative biomass and grain yield.

A modular steroid-inducible gene expression system for use in rice.

BACKGROUND: Chemically inducible systems that provide both spatial and temporal control of gene expression are essential tools, with many applications in plant biology, yet they have not been extensively tested in monocotyledonous species. RESULTS: Using Golden Gate modular cloning, we have created a monocot-optimized dexamethasone (DEX)-inducible pOp6/LhGR system and tested its efficacy in rice using the reporter enzyme ß-glucuronidase (GUS). The system is tightly regulated and highly sensitive to DEX application, with 6 h of induction sufficient to induce high levels of GUS activity in transgenic callus. In seedlings, GUS activity was detectable in the root after in vitro application of just 0.01 µM DEX. However, transgenic plants manifested severe developmental perturbations when grown on higher concentrations of DEX. The direct cause of these growth defects is not known, but the rice genome contains sequences with high similarity to the LhGR target sequence lacO, suggesting non-specific activation of endogenous genes by DEX induction. These off-target effects can be minimized by quenching with isopropyl ß-D-1-thiogalactopyranoside (IPTG). CONCLUSIONS: Our results demonstrate that the system is suitable for general use in rice, when the method of DEX application and relevant controls are tailored appropriately for each specific application.

Redundant SCARECROW genes pattern distinct cell layers in roots and leaves of maize.

The highly efficient C4 photosynthetic pathway is facilitated by 'Kranz' leaf anatomy. In Kranz leaves, closely spaced veins are encircled by concentric layers of photosynthetic bundle sheath (inner) and mesophyll (outer) cells. Here, we demonstrate that, in the C4 monocot maize, Kranz patterning is regulated by redundant function of SCARECROW 1 (ZmSCR1) and a previously uncharacterized homeologue: ZmSCR1h. ZmSCR1 and ZmSCR1h transcripts accumulate in ground meristem cells of developing leaf primordia and in Zmscr1;Zmscr1h mutant leaves, most veins are separated by one rather than two mesophyll cells; many veins have sclerenchyma above and/or below instead of mesophyll cells; and supernumerary bundle sheath cells develop. The mutant defects are unified by compromised mesophyll cell development. In addition to Kranz defects, Zmscr1;Zmscr1h mutants fail to form an organized endodermal layer in the root. Collectively, these data indicate that ZmSCR1 and ZmSCR1h redundantly regulate cell-type patterning in both the leaves and roots of maize. Leaf and root pathways are distinguished, however, by the cell layer in which they operate - mesophyll at a two-cell distance from leaf veins versus endodermis immediately adjacent to root vasculature.

LEAFY maintains apical stem cell activity during shoot development in the fern Ceratopteris richardii.

During land plant evolution, determinate spore-bearing axes (retained in extant bryophytes such as mosses) were progressively transformed into indeterminate branching shoots with specialized reproductive axes that form flowers. The LEAFY transcription factor, which is required for the first zygotic cell division in mosses and primarily for floral meristem identity in flowering plants, may have facilitated developmental innovations during these transitions. Mapping the LEAFY evolutionary trajectory has been challenging, however, because there is no functional overlap between mosses and flowering plants, and no functional data from intervening lineages. Here, we report a transgenic analysis in the fern Ceratopteris richardii that reveals a role for LEAFY in maintaining cell divisions in the apical stem cells of both haploid and diploid phases of the lifecycle. These results support an evolutionary trajectory in which an ancestral LEAFY module that promotes cell proliferation was progressively co-opted, adapted and specialized as novel shoot developmental contexts emerged.