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.

Molecular mechanisms underlying leaf development, morphological diversification, and beyond.

The basic mechanisms of leaf development have been revealed through a combination of genetics and intense analyses in select model species. The genetic basis for diversity in leaf morphology seen in nature is also being unraveled through recent advances in techniques and technologies related to genomics and transcriptomics, which have had a major impact on these comparative studies. However, this has led to the emergence of new unresolved questions about the mechanisms that generate the diversity of leaf form. Here, we provide a review of the current knowledge of the fundamental molecular genetic mechanisms underlying leaf development with an emphasis on natural variation and conserved gene regulatory networks involved in leaf development. Beyond that, we discuss open questions/enigmas in the area of leaf development, how recent technologies can best be deployed to generate a unified understanding of leaf diversity and its evolution, and what untapped fields lie ahead.

Spatial transcriptional signatures define margin morphogenesis along the proximal-distal and medio-lateral axes in tomato (Solanum lycopersicum) leaves.

Leaf morphogenesis involves cell division, expansion, and differentiation in the developing leaf, which take place at different rates and at different positions along the medio-lateral and proximal-distal leaf axes. The gene expression changes that control cell fate along these axes remain elusive due to difficulties in precisely isolating tissues. Here, we combined rigorous early leaf characterization, laser capture microdissection, and transcriptomic sequencing to ask how gene expression patterns regulate early leaf morphogenesis in wild-type tomato (Solanum lycopersicum) and the leaf morphogenesis mutant trifoliate. We observed transcriptional regulation of cell differentiation along the proximal-distal axis and identified molecular signatures delineating the classically defined marginal meristem/blastozone region during early leaf development. We describe the role of endoreduplication during leaf development, when and where leaf cells first achieve photosynthetic competency, and the regulation of auxin transport and signaling along the leaf axes. Knockout mutants of BLADE-ON-PETIOLE2 exhibited ectopic shoot apical meristem formation on leaves, highlighting the role of this gene in regulating margin tissue identity. We mapped gene expression signatures in specific leaf domains and evaluated the role of each domain in conferring indeterminacy and permitting blade outgrowth. Finally, we generated a global gene expression atlas of the early developing compound leaf.

Heinz-resistant tomato cultivars exhibit a lignin-based resistance to field dodder (Cuscuta campestris) parasitism.

Cuscuta species (dodders) are agriculturally destructive, parasitic angiosperms. These parasitic plants use haustoria as physiological bridges to extract nutrients and water from hosts. Cuscuta campestris has a broad host range and wide geographical distribution. While some wild tomato relatives are resistant, cultivated tomatoes are generally susceptible to C. campestris infestations. However, some specific Heinz tomato (Solanum lycopersicum) hybrid cultivars exhibit resistance to dodders in the field, but their defense mechanism was previously unknown. Here, we discovered that the stem cortex in these resistant lines responds with local lignification upon C. campestris attachment, preventing parasite entry into the host. Lignin Induction Factor 1 (LIF1, an AP2-like transcription factor), SlMYB55, and Cuscuta R-gene for Lignin-based Resistance 1, a CC-NBS-LRR (CuRLR1) are identified as factors that confer host resistance by regulating lignification. SlWRKY16 is upregulated upon C. campestris infestation and potentially negatively regulates LIF1 function. Intriguingly, CuRLR1 may play a role in signaling or function as an intracellular receptor for receiving Cuscuta signals or effectors, thereby regulating lignification-based resistance. In summary, these four regulators control the lignin-based resistance response in specific Heinz tomato cultivars, preventing C. campestris from parasitizing resistant tomatoes. This discovery provides a foundation for investigating multilayer resistance against Cuscuta species and has potential for application in other essential crops attacked by parasitic plants.

LATERAL ORGAN BOUNDARIES DOMAIN 25 functions as a key regulator of haustorium development in dodders.

Parasitic plants reduce crop yield worldwide. Dodder (Cuscuta campestris) is a stem parasite that attaches to its host, using haustoria to extract nutrients and water. We analyzed the transcriptome of six C. campestris tissues and identified a key gene, LATERAL ORGAN BOUNDARIES DOMAIN 25 (CcLBD25), as highly expressed in prehaustoria and haustoria. Gene coexpression networks from different tissue types and laser-capture microdissection RNA-sequencing data indicated that CcLBD25 could be essential for regulating cell wall loosening and organogenesis. We employed host-induced gene silencing by generating transgenic tomato (Solanum lycopersicum) hosts that express hairpin RNAs to target and down-regulate CcLBD25 in the parasite. Our results showed that C. campestris growing on CcLBD25 RNAi transgenic tomatoes transited to the flowering stage earlier and had reduced biomass compared with C. campestris growing on wild-type (WT) hosts, suggesting that parasites growing on transgenic plants were stressed due to insufficient nutrient acquisition. We developed an in vitro haustorium system to assay the number of prehaustoria produced on strands from C. campestris. Cuscuta campestris grown on CcLBD25 RNAi tomatoes produced fewer prehaustoria than those grown on WT tomatoes, indicating that down-regulating CcLBD25 may affect haustorium initiation. Cuscuta campestris haustoria growing on CcLBD25 RNAi tomatoes exhibited reduced pectin digestion and lacked searching hyphae, which interfered with haustorium penetration and formation of vascular connections. The results of this study elucidate the role of CcLBD25 in haustorium development and might contribute to developing parasite-resistant crops.

Leaf shape is a predictor of fruit quality and cultivar performance in tomato.

Commercial tomato (Solanum lycopersicum) is one of the most widely grown vegetable crops worldwide. Heirloom tomatoes retain extensive genetic diversity and a considerable range of fruit quality and leaf morphological traits. Here the role of leaf morphology was investigated for its impact on fruit quality. Heirloom cultivars were grown in field conditions, and BRIX by yield (BY) and other traits were measured over a 14-wk period. The complex relationships among these morphological and physiological traits were evaluated using partial least-squares path modeling, and a consensus model was developed. Photosynthesis contributed strongly to vegetative biomass and sugar content of fruits but had a negative impact on yield. Conversely leaf shape, specifically rounder leaves, had a strong positive impact on both fruit sugar content and yield. Cultivars such as Stupice and Glacier, with very round leaves, had the highest performance in both fruit sugar and yield. Our model accurately predicted BY for two commercial cultivars using leaf shape data as input. This study revealed the importance of leaf shape to fruit quality in tomato, with rounder leaves having significantly improved fruit quality. This correlation was maintained across a range of diverse genetic backgrounds and shows the importance of leaf morphology in tomato crop improvement.

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.

The molecular control of tendril development in angiosperms.

The climbing habit has evolved multiple times during the evolutionary history of angiosperms. Plants evolved various strategies for climbing, such as twining stems, tendrils and hooks. Tendrils are threadlike organs with the ability to twine around other structures through helical growth; they may be derived from a variety of structures, such as branches, leaflets and inflorescences. The genetic capacity to grow as a tendrilled climber existed in some of the earliest land plants; however, the underlying molecular basis of tendril development has been studied in only a few taxa. Here, we summarize what is known about the molecular basis of tendril development in model and candidate model species from key tendrilled families, that is, Fabaceae, Vitaceae, Cucurbitaceae, Passifloraceae and Bignoniaceae. Studies on tendril molecular genetics and development show the molecular basis of tendril formation and ontogenesis is diverse, even when tendrils have the same ontogenetic origin, for example leaflet-derived tendrils in Fabaceae and Bignoniaceae. Interestingly, all tendrils perform helical growth during contact-induced coiling, indicating that such ability is not correlated with their ontogenetic origin or phylogenetic history. Whether the same genetic networks are involved during helical growth in diverse tendrils still remains to be investigated.

Decoding the gene coexpression network underlying the ability of Gevuina avellana to live in diverse light conditions.

Gevuina avellana (Proteaceae) is a typical tree from the South American temperate rainforest. Although this species mostly regenerates in shaded understories, it exhibits an exceptional ecological breadth, being able to live under a wide range of light conditions. Here we studied the genetic basis that underlies physiological acclimation of the photosynthetic responses of G. avellana under contrasting light conditions. We analyzed carbon assimilation and light energy used for photochemical processes in plants acclimated to contrasting light conditions. Also, we used a transcriptional profile of leaf primordia from G. avellana saplings growing under different light environments in their natural habitat, to identify the gene coexpression network underpinning photosynthetic performance and light-related processes. The photosynthetic parameters revealed optimal performance regardless of light conditions. Strikingly, the mechanism involved in dissipation of excess light energy showed no significant differences between high- and low-light-acclimated plants. The gene coexpression network defined a community structure consistent with the photochemical responses, including genes involved mainly in assembly and functioning of photosystems, photoprotection, and retrograde signaling. This ecophysiological genomics approach improves our understanding of the intraspecific variability that allows G. avellana to have optimal photochemical and photoprotective mechanisms in the diverse light habitats it encounters in nature.

An intracellular transcriptomic atlas of the giant coenocyte Caulerpa taxifolia.

Convergent morphologies have arisen in plants multiple times. In non-vascular and vascular land plants, convergent morphology in the form of roots, stems, and leaves arose. The morphology of some green algae includes an anchoring holdfast, stipe, and leaf-like fronds. Such morphology occurs in the absence of multicellularity in the siphonous algae, which are single cells. Morphogenesis is separate from cellular division in the land plants, which although are multicellular, have been argued to exhibit properties similar to single celled organisms. Within the single, macroscopic cell of a siphonous alga, how are transcripts partitioned, and what can this tell us about the development of similar convergent structures in land plants? Here, we present a de novo assembled, intracellular transcriptomic atlas for the giant coenocyte Caulerpa taxifolia. Transcripts show a global, basal-apical pattern of distribution from the holdfast to the frond apex in which transcript identities roughly follow the flow of genetic information in the cell, transcription-to-translation. The analysis of the intersection of transcriptomic atlases of a land plant and Caulerpa suggests the recurrent recruitment of transcript accumulation patterns to organs over large evolutionary distances. Our results not only provide an intracellular atlas of transcript localization, but also demonstrate the contribution of transcript partitioning to morphology, independent from multicellularity, in plants.

A sister of PIN1 gene in tomato (Solanum lycopersicum) defines leaf and flower organ initiation patterns by maintaining epidermal auxin flux.

The spatiotemporal localization of the plant hormone auxin acts as a positional cue during early leaf and flower organogenesis. One of the main contributors to auxin localization is the auxin efflux carrier PIN-FORMED1 (PIN1). Phylogenetic analysis has revealed that PIN1 genes are split into two sister clades; PIN1 and the relatively uncharacterized Sister-Of-PIN1 (SoPIN1). In this paper we identify entire-2 as a loss-of-function SlSoPIN1a (Solyc10g078370) mutant in Solanum lycopersicum. The entire-2 plants are unable to specify proper leaf initiation leading to a frequent switch from the wild type spiral phyllotactic pattern to distichous and decussate patterns. Leaves in entire-2 are large and less complex and the leaflets display spatial deformities in lamina expansion, vascular development, and margin specification. During sympodial growth in entire-2 the specification of organ position and identity is greatly affected resulting in variable branching patterns on the main sympodial and inflorescence axes. To understand how SlSoPIN1a functions in establishing proper auxin maxima we used the auxin signaling reporter DR5: Venus to visualize differences in auxin localization between entire-2 and wild type. DR5: Venus visualization shows a widening of auxin localization which spreads to subepidermal tissue layers during early leaf and flower organogenesis, showing that SoPIN1 functions to focus auxin signaling to the epidermal layer. The striking spatial deformities observed in entire-2 help provide a mechanistic framework for explaining the function of the SoPIN1 clade in S.lycopersicum.

eQTL Regulating Transcript Levels Associated with Diverse Biological Processes in Tomato.

Variation in gene expression, in addition to sequence polymorphisms, is known to influence developmental, physiological, and metabolic traits in plants. Genetic mapping populations have facilitated identification of expression quantitative trait loci (eQTL), the genetic determinants of variation in gene expression patterns. We used an introgression population developed from the wild desert-adapted Solanum pennellii and domesticated tomato (Solanum lycopersicum) to identify the genetic basis of transcript level variation. We established the effect of each introgression on the transcriptome and identified approximately 7,200 eQTL regulating the steady-state transcript levels of 5,300 genes. Barnes-Hut t-distributed stochastic neighbor embedding clustering identified 42 modules revealing novel associations between transcript level patterns and biological processes. The results showed a complex genetic architecture of global transcript abundance pattern in tomato. Several genetic hot spots regulating a large number of transcript level patterns relating to diverse biological processes such as plant defense and photosynthesis were identified. Important eQTL regulating transcript level patterns were related to leaf number and complexity as well as hypocotyl length. Genes associated with leaf development showed an inverse correlation with photosynthetic gene expression, but eQTL regulating genes associated with leaf development and photosynthesis were dispersed across the genome. This comprehensive eQTL analysis details the influence of these loci on plant phenotypes and will be a valuable community resource for investigations on the genetic effects of eQTL on phenotypic traits in tomato.

Resolving distinct genetic regulators of tomato leaf shape within a heteroblastic and ontogenetic context.

Leaf shape is mutable, changing in ways modulated by both development and environment within genotypes. A complete model of leaf phenotype would incorporate the changes in leaf shape during juvenile-to-adult phase transitions and the ontogeny of each leaf. Here, we provide a morphometric description of >33,000 leaflets from a set of tomato (Solanum spp) introgression lines grown under controlled environment conditions. We first compare the shape of these leaves, arising during vegetative development, with >11,000 previously published leaflets from a field setting and >11,000 leaflets from wild tomato relatives. We then quantify the changes in shape, across ontogeny, for successive leaves in the heteroblastic series. Using principal component analysis, we then separate genetic effects modulating (1) the overall shape of all leaves versus (2) the shape of specific leaves in the series, finding the former more heritable than the latter and comparing quantitative trait loci regulating each. Our results demonstrate that phenotype is highly contextual and that unbiased assessments of phenotype, for quantitative genetic or other purposes, would ideally sample the many developmental and environmental factors that modulate it.

Evolutionary developmental transcriptomics reveals a gene network module regulating interspecific diversity in plant leaf shape.

Despite a long-standing interest in the genetic basis of morphological diversity, the molecular mechanisms that give rise to developmental variation are incompletely understood. Here, we use comparative transcriptomics coupled with the construction of gene coexpression networks to predict a gene regulatory network (GRN) for leaf development in tomato and two related wild species with strikingly different leaf morphologies. The core network in the leaf developmental GRN contains regulators of leaf morphology that function in global cell proliferation with peripheral gene network modules (GNMs). The BLADE-ON-PETIOLE (BOP) transcription factor in one GNM controls the core network by altering effective concentration of the KNOTTED-like HOMEOBOX gene product. Comparative network analysis and experimental perturbations of BOP levels suggest that variation in BOP expression could explain the diversity in leaf complexity among these species through dynamic rewiring of interactions in the GRN. The peripheral location of the BOP-containing GNM in the leaf developmental GRN and the phenotypic mimics of evolutionary diversity caused by alteration in BOP levels identify a key role for this GNM in canalizing the leaf morphospace by modifying the maturation schedule of leaves to create morphological diversity.

Transcriptomic analysis suggests a key role for SQUAMOSA PROMOTER BINDING PROTEIN LIKE, NAC and YUCCA genes in the heteroblastic development of the temperate rainforest tree Gevuina avellana (Proteaceae).

Heteroblasty, the temporal development of the meristem, can produce diverse leaf shapes within a plant. Gevuina avellana, a tree from the South American temperate rainforest shows strong heteroblasty affecting leaf shape, transitioning from juvenile simple leaves to highly pinnate adult leaves. Light availability within the forest canopy also modulates its leaf size and complexity. Here we studied how the interaction between the light environment and the heteroblastic progression of leaves is coordinated in this species. We used RNA-seq on the Illumina platform to compare the range of transcriptional responses in leaf primordia of G. avellana at different heteroblastic stages and growing under different light environments. We found a steady up-regulation of SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL), NAC, YUCCA and AGAMOUS-LIKE genes associated with increases in age, leaf complexity, and light availability. In contrast, expression of TCP, TPR and KNOTTED1 homeobox genes showed a sustained down-regulation. Additionally, genes involved in auxin synthesis/transport and jasmonate activity were differentially expressed, indicating an active regulation of processes controlled by these hormones. Our large-scale transcriptional analysis of the leaf primordia of G. avellana sheds light on the integration of internal and external cues during heteroblastic development in this species.

Light-Induced Indeterminacy Alters Shade-Avoiding Tomato Leaf Morphology.

Plants sense the foliar shade of competitors and alter their developmental programs through the shade-avoidance response. Internode and petiole elongation, and changes in overall leaf area and leaf mass per area, are the stereotypical architectural responses to foliar shade in the shoot. However, changes in leaf shape and complexity in response to shade remain incompletely, and qualitatively, described. Using a meta-analysis of more than 18,000 previously published leaflet outlines, we demonstrate that shade avoidance alters leaf shape in domesticated tomato (Solanum lycopersicum) and wild relatives. The effects of shade avoidance on leaf shape are subtle with respect to individual traits but are combinatorially strong. We then seek to describe the developmental origins of shade-induced changes in leaf shape by swapping plants between light treatments. Leaf size is light responsive late into development, but patterning events, such as stomatal index, are irrevocably specified earlier. Observing that shade induces increases in shoot apical meristem size, we then describe gene expression changes in early leaf primordia and the meristem using laser microdissection. We find that in leaf primordia, shade avoidance is not mediated through canonical pathways described in mature organs but rather through the expression of KNOTTED1-LIKE HOMEOBOX and other indeterminacy genes, altering known developmental pathways responsible for patterning leaf shape. We also demonstrate that shade-induced changes in leaf primordium gene expression largely do not overlap with those found in successively initiated leaf primordia, providing evidence against classic hypotheses that shaded leaf morphology results from the prolonged production of juvenile leaf types.

A quantitative genetic basis for leaf morphology in a set of precisely defined tomato introgression lines.

Introgression lines (ILs), in which genetic material from wild tomato species is introgressed into a domesticated background, have been used extensively in tomato (Solanum lycopersicum) improvement. Here, we genotype an IL population derived from the wild desert tomato Solanum pennellii at ultrahigh density, providing the exact gene content harbored by each line. To take advantage of this information, we determine IL phenotypes for a suite of vegetative traits, ranging from leaf complexity, shape, and size to cellular traits, such as stomatal density and epidermal cell phenotypes. Elliptical Fourier descriptors on leaflet outlines provide a global analysis of highly heritable, intricate aspects of leaf morphology. We also demonstrate constraints between leaflet size and leaf complexity, pavement cell size, and stomatal density and show independent segregation of traits previously assumed to be genetically coregulated. Meta-analysis of previously measured traits in the ILs shows an unexpected relationship between leaf morphology and fruit sugar levels, which RNA-Seq data suggest may be attributable to genetically coregulated changes in fruit morphology or the impact of leaf shape on photosynthesis. Together, our results both improve upon the utility of an important genetic resource and attest to a complex, genetic basis for differences in leaf morphology between natural populations.

Trifoliate encodes an MYB transcription factor that modulates leaf and shoot architecture in tomato.

Leaf morphology and the pattern of shoot branching determine to a large extent the growth habit of seed plants. Until recently, the developmental processes that led to the establishment of these morphological structures seemed unrelated. Here, we show that the tomato Trifoliate (Tf) gene plays a crucial role in both processes, affecting the formation of leaflets in the compound tomato leaf and the initiation of axillary meristems in the leaf axil. Tf encodes a myeloblastosis oncoprotein (MYB)-like transcription factor related to the Arabidopsis thaliana LATERAL ORGAN FUSION1 (LOF1) and LOF2 proteins. Tf is expressed in the leaf margin, where leaflets are formed, and in the leaf axil, where axillary meristems initiate. During tomato ontogeny, expression of Tf in young leaf primordia increases, correlating with a rise in leaf dissection (heteroblasty). Formation of leaflets and initiation of axillary meristems can be traced back to groups of pluripotent cells. Tf function is required to inhibit differentiation of these cells and thereby to maintain their morphogenetic competence, a fundamental process in plant development. KNOTTED1-LIKE proteins, which are known regulators in tomato leaf dissection, require Tf activity to exert their function in the basal part of the leaf. Similarly, the plant hormone auxin needs Tf activity to initiate the formation of lateral leaflets. Thus, leaf dissection and shoot branching rely on a conserved mechanism that regulates the morphogenetic competence of cells at the leaf margin and in the leaf axil.