Innovation, conservation, and repurposing of gene function in root cell type development.

Plant species have evolved myriads of solutions, including complex cell type development and regulation, to adapt to dynamic environments. To understand this cellular diversity, we profiled tomato root cell type translatomes. Using xylem differentiation in tomato, examples of functional innovation, repurposing, and conservation of transcription factors are described, relative to the model plant Arabidopsis. Repurposing and innovation of genes are further observed within an exodermis regulatory network and illustrate its function. Comparative translatome analyses of rice, tomato, and Arabidopsis cell populations suggest increased expression conservation of root meristems compared with other homologous populations. In addition, the functions of constitutively expressed genes are more conserved than those of cell type/tissue-enriched genes. These observations suggest that higher order properties of cell type and pan-cell type regulation are evolutionarily conserved between plants and animals.

ggPlantmap: an open-source R package for the creation of informative and quantitative ggplot maps derived from plant images.

As plant research generates an ever-growing volume of spatial quantitative data, the need for decentralized and user-friendly visualization tools to explore large and complex datasets tools becomes crucial. Existing resources, such as the Plant eFP (electronic Fluorescent Pictograph) viewer, have played a pivotal role on the communication of gene expression data across many plant species. However, although widely used by the plant research community, the Plant eFP viewer lacks open and user-friendly tools for the creation of customized expression maps independently. Plant biologists with less coding experience can often encounter challenges when attempting to explore ways to communicate their own spatial quantitative data. We present 'ggPlantmap' an open-source R package designed to address this challenge by providing an easy and user-friendly method for the creation of ggplot representative maps from plant images. ggPlantmap is built in R, one of the most used languages in biology to empower plant scientists to create and customize eFP-like viewers tailored to their experimental data. Here, we provide an overview of the package and tutorials that are accessible even to users with minimal R programming experience. We hope that ggPlantmap can assist the plant science community, fostering innovation and improving our understanding of plant development and function.

Mechanisms of far-red light-mediated dampening of defense against Botrytis cinerea in tomato leaves.

Plants detect neighboring competitors through a decrease in the ratio between red and far-red light (R:FR). This decreased R:FR is perceived by phytochrome photoreceptors and triggers shade avoidance responses such as shoot elongation and upward leaf movement (hyponasty). In addition to promoting elongation growth, low R:FR perception enhances plant susceptibility to pathogens: the growth-defense tradeoff. Although increased susceptibility in low R:FR has been studied for over a decade, the associated timing of molecular events is still unknown. Here, we studied the chronology of FR-induced susceptibility events in tomato (Solanum lycopersicum) plants pre-exposed to either white light (WL) or WL supplemented with FR light (WL+FR) prior to inoculation with the necrotrophic fungus Botrytis cinerea (B.c.). We monitored the leaf transcriptional changes over a 30-h time course upon infection and followed up with functional studies to identify mechanisms. We found that FR-induced susceptibility in tomato is linked to a general dampening of B.c.-responsive gene expression, and a delay in both pathogen recognition and jasmonic acid-mediated defense gene expression. In addition, we found that the supplemental FR-induced ethylene emissions affected plant immune responses under the WL+FR condition. This study improves our understanding of the growth-immunity tradeoff, while simultaneously providing leads to improve tomato resistance against pathogens in dense cropping systems.

Translational regulation contributes to the elevated CO2 response in two Solanum species.

Understanding the impact of elevated CO2 (eCO2 ) in global agriculture is important given climate change projections. Breeding climate-resilient crops depends on genetic variation within naturally varying populations. The effect of genetic variation in response to eCO2 is poorly understood, especially in crop species. We describe the different ways in which Solanum lycopersicum and its wild relative S. pennellii respond to eCO2 , from cell anatomy, to the transcriptome, and metabolome. We further validate the importance of translational regulation as a potential mechanism for plants to adaptively respond to rising levels of atmospheric CO2 .

Convergent evolution of gene regulatory networks underlying plant adaptations to dry environments.

Plants transitioned from an aquatic to a terrestrial lifestyle during their evolution. On land, fluctuations on water availability in the environment became one of the major problems they encountered. The appearance of morpho-physiological adaptations to cope with and tolerate water loss from the cells was undeniably useful to survive on dry land. Some of these adaptations, such as carbon concentrating mechanisms (CCMs), desiccation tolerance (DT) and root impermeabilization, appeared in multiple plant lineages. Despite being crucial for evolution on land, it has been unclear how these adaptations convergently evolved in the various plant lineages. Recent advances on whole genome and transcriptome sequencing are revealing that co-option of genes and gene regulatory networks (GRNs) is a common feature underlying the convergent evolution of these adaptations. In this review, we address how the study of CCMs and DT has provided insight into convergent evolution of GRNs underlying plant adaptation to dry environments, and how these insights could be applied to currently emerging understanding of evolution of root impermeabilization through different barrier cell types. We discuss examples of co-option, conservation and innovation of genes and GRNs at the cell, tissue and organ levels revealed by recent phylogenomic (comparative genomic) and comparative transcriptomic studies.

Profiling of Accessible Chromatin Regions across Multiple Plant Species and Cell Types Reveals Common Gene Regulatory Principles and New Control Modules.

The transcriptional regulatory structure of plant genomes remains poorly defined relative to animals. It is unclear how many cis-regulatory elements exist, where these elements lie relative to promoters, and how these features are conserved across plant species. We employed the assay for transposase-accessible chromatin (ATAC-seq) in four plant species (Arabidopsis thaliana, Medicago truncatula, Solanum lycopersicum, and Oryza sativa) to delineate open chromatin regions and transcription factor (TF) binding sites across each genome. Despite 10-fold variation in intergenic space among species, the majority of open chromatin regions lie within 3 kb upstream of a transcription start site in all species. We find a common set of four TFs that appear to regulate conserved gene sets in the root tips of all four species, suggesting that TF-gene networks are generally conserved. Comparative ATAC-seq profiling of Arabidopsis root hair and non-hair cell types revealed extensive similarity as well as many cell-type-specific differences. Analyzing TF binding sites in differentially accessible regions identified a MYB-driven regulatory module unique to the hair cell, which appears to control both cell fate regulators and abiotic stress responses. Our analyses revealed common regulatory principles among species and shed light on the mechanisms producing cell-type-specific transcriptomes during development.

Far-red light promotes Botrytis cinerea disease development in tomato leaves via jasmonate-dependent modulation of soluble sugars.

Plants experience a decrease in the red:far-red light ratio (R:FR) when grown at high planting density. In addition to eliciting the shade avoidance response, low R:FR also enhances plant susceptibility to pathogens via modulation of defense hormone-mediated responses. However, other mechanisms, also affected by low R:FR, have not been considered as potential components in FR-induced susceptibility. Here, we identify FR-induced accumulation of leaf soluble sugars as a novel component of FR-induced susceptibility. We observed that phytochrome inactivation by FR or phytochrome B mutation was associated with elevated leaf glucose and fructose levels and enhanced disease severity caused by Botrytis cinerea. By experimentally manipulating internal leaf sugar levels, we found that the FR-induced susceptibility in tomato was partly sugar-dependent. Further analysis revealed that the observed sugar accumulation in supplemental FR occurred in a jasmonic acid (JA)-dependent manner, and the JA biosynthesis mutant def1 also displayed elevated soluble sugar levels, which was rescued by exogenous methyl jasmonate (MeJA) application. We propose that the reduced JA responsiveness under low R:FR promotes disease symptoms not only via dampened induction of defense responses, but also via increased levels of soluble sugars that supports pathogen growth in tomato leaves.

Nuclear Transcriptomes at High Resolution Using Retooled INTACT.

Isolated nuclei provide access to early steps in gene regulation involving chromatin as well as transcript production and processing. Here, we describe transfer of the isolation of nuclei from tagged specific cell types (INTACT) to the monocot rice (Oryza sativa L.). The purification of biotinylated nuclei was redesigned by replacing the outer nuclear-envelope-targeting domain of the nuclear tagging fusion (NTF) protein with an outer nuclear-envelope-anchored domain. This modified NTF was combined with codon-optimized Escherichia coli BirA in a single T-DNA construct. We also developed inexpensive methods for INTACT, T-DNA insertion mapping, and profiling of the complete nuclear transcriptome, including a ribosomal RNA degradation procedure that minimizes pre-ribosomal RNA (pre-rRNA) transcripts. A high-resolution comparison of nuclear and steady-state poly(A)+ transcript populations of seedling root tips confirmed the capture of pre-messenger RNA (pre-mRNA) and exposed distinctions in diversity and abundance of the nuclear and total transcriptomes. This retooled INTACT can enable high-resolution monitoring of the nuclear transcriptome and chromatin in specific cell types of rice and other species.

DNA methylation and gene expression regulation associated with vascularization in Sorghum bicolor.

Plant secondary cell walls constitute the majority of plant biomass. They are predominantly found in xylem cells, which are derived from vascular initials during vascularization. Little is known about these processes in grass species despite their emerging importance as biomass feedstocks. The targeted biofuel crop Sorghum bicolor has a sequenced and well-annotated genome, making it an ideal monocot model for addressing vascularization and biomass deposition. Here we generated tissue-specific transcriptome and DNA methylome data from sorghum shoots, roots and developing root vascular and nonvascular tissues. Many genes associated with vascular development in other species show enriched expression in developing vasculature. However, several transcription factor families varied in vascular expression in sorghum compared with Arabidopsis and maize. Furthermore, differential expression of genes associated with DNA methylation were identified between vascular and nonvascular tissues, implying that changes in DNA methylation are a feature of sorghum root vascularization, which we confirmed using tissue-specific DNA methylome data. Roots treated with a DNA methylation inhibitor also showed a significant decrease in root length. Tissues and organs can be discriminated based on their genomic methylation patterns and methylation context. Consequently, tissue-specific changes in DNA methylation are part of the normal developmental process.

Hairy root transformation using Agrobacterium rhizogenes as a tool for exploring cell type-specific gene expression and function using tomato as a model.

Agrobacterium rhizogenes (or Rhizobium rhizogenes) is able to transform plant genomes and induce the production of hairy roots. We describe the use of A. rhizogenes in tomato (Solanum spp.) to rapidly assess gene expression and function. Gene expression of reporters is indistinguishable in plants transformed by Agrobacterium tumefaciens as compared with A. rhizogenes. A root cell type- and tissue-specific promoter resource has been generated for domesticated and wild tomato (Solanum lycopersicum and Solanum pennellii, respectively) using these approaches. Imaging of tomato roots using A. rhizogenes coupled with laser scanning confocal microscopy is facilitated by the use of a membrane-tagged protein fused to a red fluorescent protein marker present in binary vectors. Tomato-optimized isolation of nuclei tagged in specific cell types and translating ribosome affinity purification binary vectors were generated and used to monitor associated messenger RNA abundance or chromatin modification. Finally, transcriptional reporters, translational reporters, and clustered regularly interspaced short palindromic repeats-associated nuclease9 genome editing demonstrate that SHORT-ROOT and SCARECROW gene function is conserved between Arabidopsis (Arabidopsis thaliana) and tomato.

Omics and modelling approaches for understanding regulation of asymmetric cell divisions in arabidopsis and other angiosperm plants.

BACKGROUND: Asymmetric cell divisions are formative divisions that generate daughter cells of distinct identity. These divisions are coordinated by either extrinsic ('niche-controlled') or intrinsic regulatory mechanisms and are fundamentally important in plant development. SCOPE: This review describes how asymmetric cell divisions are regulated during development and in different cell types in both the root and the shoot of plants. It further highlights ways in which omics and modelling approaches have been used to elucidate these regulatory mechanisms. For example, the regulation of embryonic asymmetric divisions is described, including the first divisions of the zygote, formative vascular divisions and divisions that give rise to the root stem cell niche. Asymmetric divisions of the root cortex endodermis initial, pericycle cells that give rise to the lateral root primordium, procambium, cambium and stomatal cells are also discussed. Finally, a perspective is provided regarding the role of other hormones or regulatory molecules in asymmetric divisions, the presence of segregated determinants and the usefulness of modelling approaches in understanding network dynamics within these very special cells. CONCLUSIONS: Asymmetric cell divisions define plant development. High-throughput genomic and modelling approaches can elucidate their regulation, which in turn could enable the engineering of plant traits such as stomatal density, lateral root development and wood formation.

A suberized exodermis is required for tomato drought tolerance.

Plant roots integrate environmental signals with development using exquisite spatiotemporal control. This is apparent in the deposition of suberin, an apoplastic diffusion barrier, which regulates flow of water, solutes and gases, and is environmentally plastic. Suberin is considered a hallmark of endodermal differentiation but is absent in the tomato endodermis. Instead, suberin is present in the exodermis, a cell type that is absent in the model organism Arabidopsis thaliana. Here we demonstrate that the suberin regulatory network has the same parts driving suberin production in the tomato exodermis and the Arabidopsis endodermis. Despite this co-option of network components, the network has undergone rewiring to drive distinct spatial expression and with distinct contributions of specific genes. Functional genetic analyses of the tomato MYB92 transcription factor and ASFT enzyme demonstrate the importance of exodermal suberin for a plant water-deficit response and that the exodermal barrier serves an equivalent function to that of the endodermis and can act in its place.

Multiple Arabidopsis genes primed for recruitment into C4 photosynthesis.

C(4) photosynthesis occurs in the most productive crops and vegetation on the planet, and has become widespread because it allows increased rates of photosynthesis compared with the ancestral C(3) pathway. Leaves of C(4) plants typically possess complicated alterations to photosynthesis, such that its reactions are compartmented between mesophyll and bundle sheath cells. Despite its complexity, the C(4) pathway has arisen independently in 62 separate lineages of land plants, and so represents one of the most striking examples of convergent evolution known. We demonstrate that elements in untranslated regions (UTRs) of multiple genes important for C(4) photosynthesis contribute to the metabolic compartmentalization characteristic of a C(4) leaf. Either the 5' or the 3' UTR is sufficient for cell specificity, indicating that functional redundancy underlies this key aspect of C(4) gene expression. Furthermore, we show that orthologous PPDK and CA genes from the C(3) plant Arabidopsis thaliana are primed for recruitment into the C(4) pathway. Elements sufficient for M-cell specificity in C(4) leaves are also present in both the 5' and 3' UTRs of these C(3) A. thaliana genes. These data indicate functional latency within the UTRs of genes from C(3) species that have been recruited into the C(4) pathway. The repeated recruitment of pre-existing cis-elements in C(3) genes may have facilitated the evolution of C(4) photosynthesis. These data also highlight the importance of alterations in trans in producing a functional C(4) leaf, and so provide insight into both the evolution and molecular basis of this important type of photosynthesis.

Why did glutamate, GABA, and melatonin become intercellular signalling molecules in plants?

Intercellular signalling is an indispensable part of multicellular life. Understanding the commonalities and differences in how signalling molecules function in two remote branches of the tree of life may shed light on the reasons these molecules were originally recruited for intercellular signalling. Here we review the plant function of three highly studied animal intercellular signalling molecules, namely glutamate, γ-aminobutyric acid (GABA), and melatonin. By considering both their signalling function in plants and their broader physiological function, we suggest that molecules with an original function as key metabolites or active participants in reactive ion species scavenging have a high chance of becoming intercellular signalling molecules. Naturally, the evolution of machinery to transduce a message across the plasma membrane is necessary. This fact is demonstrated by three other well-studied animal intercellular signalling molecules, namely serotonin, dopamine, and acetylcholine, for which there is currently no evidence that they act as intercellular signalling molecules in plants.

Mechanodetection of neighbor plants elicits adaptive leaf movements through calcium dynamics.

Plants detect their neighbors via various cues, including reflected light and touching of leaf tips, which elicit upward leaf movement (hyponasty). It is currently unknown how touch is sensed and how the signal is transferred from the leaf tip to the petiole base that drives hyponasty. Here, we show that touch-induced hyponasty involves a signal transduction pathway that is distinct from light-mediated hyponasty. We found that mechanostimulation of the leaf tip upon touching causes cytosolic calcium ([Ca2+]cyt induction in leaf tip trichomes that spreads towards the petiole. Both perturbation of the calcium response and the absence of trichomes reduce touch-induced hyponasty. Finally, using plant competition assays, we show that touch-induced hyponasty is adaptive in dense stands of Arabidopsis. We thus establish a novel, adaptive mechanism regulating hyponastic leaf movement in response to mechanostimulation by neighbors in dense vegetation.

An mRNA blueprint for C4 photosynthesis derived from comparative transcriptomics of closely related C3 and C4 species.

C(4) photosynthesis involves alterations to the biochemistry, cell biology, and development of leaves. Together, these modifications increase the efficiency of photosynthesis, and despite the apparent complexity of the pathway, it has evolved at least 45 times independently within the angiosperms. To provide insight into the extent to which gene expression is altered between C(3) and C(4) leaves, and to identify candidates associated with the C(4) pathway, we used massively parallel mRNA sequencing of closely related C(3) (Cleome spinosa) and C(4) (Cleome gynandra) species. Gene annotation was facilitated by the phylogenetic proximity of Cleome and Arabidopsis (Arabidopsis thaliana). Up to 603 transcripts differ in abundance between these C(3) and C(4) leaves. These include 17 transcription factors, putative transport proteins, as well as genes that in Arabidopsis are implicated in chloroplast movement and expansion, plasmodesmatal connectivity, and cell wall modification. These are all characteristics known to alter in a C(4) leaf but that previously had remained undefined at the molecular level. We also document large shifts in overall transcription profiles for selected functional classes. Our approach defines the extent to which transcript abundance in these C(3) and C(4) leaves differs, provides a blueprint for the NAD-malic enzyme C(4) pathway operating in a dicotyledon, and furthermore identifies potential regulators. We anticipate that comparative transcriptomics of closely related species will provide deep insight into the evolution of other complex traits.

C acid decarboxylases required for C photosynthesis are active in the mid-vein of the C species Arabidopsis thaliana, and are important in sugar and amino acid metabolism.

Cells associated with veins of petioles of C(3) tobacco possess high activities of the decarboxylase enzymes required in C(4) photosynthesis. It is not clear whether this is the case in other C(3) species, nor whether these enzymes provide precursors for specific biosynthetic pathways. Here, we investigate the activity of C(4) acid decarboxylases in the mid-vein of Arabidopsis, identify regulatory regions sufficient for this activity, and determine the impact of removing individual isoforms of each protein on mid-vein metabolite profiles. This showed that radiolabelled malate and bicarbonate fed to the xylem stream were incorporated into soluble and insoluble material in the mid-vein of Arabidopsis leaves. Compared with the leaf lamina, mid-veins possessed high activities of NADP-dependent malic enzyme (NADP-ME), NAD-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). Transcripts derived from both NAD-ME, one PCK and two of the four NADP-ME genes were detectable in these veinal cells. The promoters of each decarboxylase gene were sufficient for expression in mid-veins. Analysis of insertional mutants revealed that cytosolic NADP-ME2 is responsible for 80% of NADP-ME activity in mid-veins. Removing individual decarboxylases affected the abundance of amino acids derived from pyruvate and phosphoenolpyruvate. Reducing cytosolic NADP-ME activity preferentially affected the sugar content, whereas abolishing NAD-ME affected both the amino acid and the glucosamine content of mid-veins.

Strategies for engineering a two-celled C(4) photosynthetic pathway into rice.

Every day almost one billion people suffer from chronic hunger, and the situation is expected to deteriorate with a projected population growth to 9 billion worldwide by 2050. In order to provide adequate nutrition into the future, rice yields in Asia need to increase by 60%, a change that may be achieved by introduction of the C(4) photosynthetic cycle into rice. The international C(4) Rice Consortium was founded in order to test the feasibility of installing the C(4) engine into rice. This review provides an update on two of the many approaches employed by the C(4) Rice Consortium: namely, metabolic C(4) engineering and identification of determinants of leaf anatomy by mutant screens. The aim of the metabolic C(4) engineering approach is to generate a two-celled C(4) shuttle in rice by expressing the classical enzymes of the NADP-ME C(4) cycle in a cell-appropriate manner. The aim is also to restrict RuBisCO and glycine decarboxylase expression to the bundle sheath (BS) cells of rice in a C(4)-like fashion by specifically down-regulating their expression in rice mesophyll (M) cells. In addition to the changes in biochemistry, two-celled C(4) species show a convergence in leaf anatomy that include increased vein density and reduced numbers of M cells between veins. By screening rice activation-tagged lines and loss-of-function sorghum mutants we endeavour to identify genes controlling these key traits.

Low-cost and High-throughput RNA-seq Library Preparation for Illumina Sequencing from Plant Tissue.

Transcriptome analysis can provide clues to biological processes affected in different genetic backgrounds or/and under various conditions. The price of RNA sequencing (RNA-seq) has decreased enough so that medium- to large-scale transcriptome analyses in a range of conditions are feasible. However, the price and variety of options for library preparation of RNA-seq can still be daunting to those who would like to use RNA-seq for their first time or for a single experiment. Among the criteria for selecting a library preparation protocol are the method of RNA isolation, nucleotide fragmentation to obtain desired size range, and library indexing to pool sequencing samples for multiplexing. Here, we present a high-quality and a high-throughput option for preparing libraries from polyadenylated mRNA for transcriptome analysis. Both high-quality and high-throughput protocol options include steps of mRNA enrichment through magnetic bead-enabled precipitation of the poly-A tail, cDNA synthesis, and then fragmentation and adapter addition simultaneously through Tn5-mediated 'tagmentation'. All steps of the protocols have been validated with Arabidopsis thaliana leaf and seedling tissues and streamlined to work together, with minimal cost in money and time, thus intended to provide a beginner-friendly start-to-finish RNA-seq library preparation for transcriptome analysis.