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.

BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction.

Next Generation Sequencing (NGS) is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq) has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing the terminal breathing of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq) reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE) libraries and can easily extend to full transcript coverage shotgun (SHO) type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.

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.

Transcriptional, posttranscriptional, and posttranslational regulation of SHOOT MERISTEMLESS gene expression in Arabidopsis determines gene function in the shoot apex.

The activity of SHOOT MERISTEMLESS (STM) is required for the functioning of the shoot apical meristem (SAM). STM is expressed in the SAM but is down-regulated at the site of leaf initiation. STM is also required for the formation of compound leaves. However, how the activity of STM is regulated at the transcriptional, posttranscriptional, and posttranslational levels is poorly understood. We previously found two conserved noncoding sequences in the promoters of STM-like genes across angiosperms, the K-box and the RB-box. Here, we characterize the function of the RB-box in Arabidopsis (Arabidopsis thaliana). The RB-box, along with the K-box, regulates the expression of STM in leaf sinuses, which are areas on the leaf blade with meristematic potential. The RB-box also contributes to restrict STM expression to the SAM. We identified FAR1-RELATED SEQUENCES-RELATED FACTOR1 (FRF1) as a binding factor to the RB-box region. FRF1 is an uncharacterized member of a subfamily of four truncated proteins related to the FAR1-RELATED SEQUENCES factors. Internal deletion analysis of the STM promoter identified a region required to repress the expression of STM in hypocotyls. Expression of STM in leaf primordia under the control of the JAGGED promoter produced plants with partially undifferentiated leaves. We further found that the ELK domain has a role in the posttranslational regulation of STM by affecting the nuclear localization of STM.

Truncation of LEAFY COTYLEDON1 protein is required for asexual reproduction in Kalanchoë daigremontiana.

Kalanchoë daigremontiana reproduces asexually by generating numerous plantlets on its leaf margins. The formation of plantlets requires the somatic initiation of organogenic and embryogenic developmental programs in the leaves. However, unlike normal embryogenesis in seeds, leaf somatic embryogenesis bypasses seed dormancy to form viable plantlets. In Arabidopsis (Arabidopsis thaliana), seed dormancy and embryogenesis are initiated by the transcription factor LEAFY COTYLEDON1 (LEC1). The K. daigremontiana ortholog of LEC1 is expressed during leaf somatic embryo development. However, KdLEC1 encodes for a LEC1-type protein that has a unique B domain, with 11 unique amino acids and a premature stop codon. Moreover, the truncated KdLEC1 protein is not functional in Arabidopsis. Here, we show that K. daigremontiana transgenic plants expressing a functional, chimeric KdLEC1 gene under the control of Arabidopsis LEC1 promoter caused several developmental defects to leaf somatic embryos, including seed dormancy characteristics. The dormant plantlets also behaved as typical dormant seeds. Transgenic plantlets accumulated oil bodies and responded to the abscisic acid biosynthesis inhibitor fluridone, which broke somatic-embryo dormancy and promoted their normal development. Our results indicate that having a mutated form of LEC1 gene in K. daigremontiana is essential to bypass dormancy in the leaf embryos and generate viable plantlets, suggesting that the loss of a functional LEC1 promotes viviparous leaf somatic embryos and thus enhances vegetative propagation in K. daigremontiana. Mutations resulting in truncated LEC1 proteins may have been of a selective advantage in creating somatic propagules, because such mutations occurred independently in several Kalanchoë species, which form plantlets constitutively.

De novo assembly and characterization of the transcriptome of the parasitic weed dodder identifies genes associated with plant parasitism.

Parasitic flowering plants are one of the most destructive agricultural pests and have major impact on crop yields throughout the world. Being dependent on finding a host plant for growth, parasitic plants penetrate their host using specialized organs called haustoria. Haustoria establish vascular connections with the host, which enable the parasite to steal nutrients and water. The underlying molecular and developmental basis of parasitism by plants is largely unknown. In order to investigate the process of parasitism, RNAs from different stages (i.e. seed, seedling, vegetative strand, prehaustoria, haustoria, and flower) were used to de novo assemble and annotate the transcriptome of the obligate plant stem parasite dodder (Cuscuta pentagona). The assembled transcriptome was used to dissect transcriptional dynamics during dodder development and parasitism and identified key gene categories involved in the process of plant parasitism. Host plant infection is accompanied by increased expression of parasite genes underlying transport and transporter categories, response to stress and stimuli, as well as genes encoding enzymes involved in cell wall modifications. By contrast, expression of photosynthetic genes is decreased in the dodder infective stages compared with normal stem. In addition, genes relating to biosynthesis, transport, and response of phytohormones, such as auxin, gibberellins, and strigolactone, were differentially expressed in the dodder infective stages compared with stems and seedlings. This analysis sheds light on the transcriptional changes that accompany plant parasitism and will aid in identifying potential gene targets for use in controlling the infestation of crops by parasitic weeds.

KNOX1 genes regulate lignin deposition and composition in monocots and dicots.

Plant secondary cell walls are deposited mostly in vascular tissues such as xylem vessels, tracheids, and fibers. These cell walls are composed of a complex matrix of compounds including cellulose, hemicellulose, and lignin. Lignin functions primarily to maintain the structural and mechanical integrity of both the transport vessel and the entire plant itself. Since lignin has been identified as a major source of biomass for biofuels, regulation of secondary cell wall biosynthesis has been a topic of much recent investigation. Biosynthesis and patterning of lignin involves many developmental and environmental cues including evolutionarily conserved transcriptional regulatory modules and hormonal signals. Here, we investigate the role of the class I Knotted1-like-homeobox (KNOX) genes and gibberellic acid in the lignin biosynthetic pathway in a representative monocot and a representative eudicot. Knotted1 overexpressing mutant plants showed a reduction in lignin content in both maize and tobacco. Expression of four key lignin biosynthesis genes was analyzed and revealed that KNOX1 genes regulate at least two steps in the lignin biosynthesis pathway. The negative regulation of lignin both in a monocot and a eudicot by the maize Kn1 gene suggests that lignin biosynthesis may be preserved across large phylogenetic distances. The evolutionary implications of regulation of lignification across divergent species are discussed.

Duplicated STM-like KNOX I genes act in floral meristem activity in Eschscholzia californica (Papaveraceae).

In angiosperms, the shoot apical meristem is at the origin of leaves and stems and is eventually transformed into the floral meristem. Class I knotted-like homeobox (KNOX I) genes are known as crucial regulators of shoot meristem formation and maintenance. KNOX I genes maintain the undifferentiated state of the apical meristem and are locally downregulated upon leaf initiation. In Arabidopsis, KNOX I genes, especially SHOOTMERISTEMLESS (STM), have been shown to regulate flower development and the formation of carpels. We investigated the role of STM-like genes in the reproductive development of Eschscholzia californica, to learn more about the evolution of KNOX I gene function in basal eudicots. We identified two orthologs of STM in Eschscholzia, EcSTM1 and EcSTM2, which are predominantly expressed in floral tissues. In contrast, a KNAT1/BP-like and a KNAT2/6-like KNOX I gene are mainly expressed in vegetative organs. Virus-induced gene silencing (VIGS) was used to knockdown gene expression, revealing that both EcSTM genes are required for the formation of reproductive organs. Silencing of EcSTM1 resulted in the loss of the gynoecium and a reduced number of stamens. EcSTM2-VIGS flowers had reduced and defective gynoecia and a stronger reduction in the number of stamen than observed in EcSTM1-VIGS. Co-silencing of both genes led to more pronounced phenotypes. In addition, silencing of EcSTM2 alone or together with EcSTM1 resulted in altered patterns of internodal elongation and sometimes in other floral defects. Our data suggest that some aspects of STM function present in Arabidopsis evolved already before the basal eudicots diverged from core eudicots.

Interspecific RNA interference of SHOOT MERISTEMLESS-like disrupts Cuscuta pentagona plant parasitism.

Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA-mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection.

A new development: evolving concepts in leaf ontogeny.

Elucidation of gene regulatory networks (GRNs) underlying aspects of leaf development in multiple model species has uncovered surprisingly plastic regulatory architecture. The meticulously mapped network interactions in one model species cannot now be assumed to map directly onto a different species. Despite these overall differences, however, many modules do appear to be almost universal. Extrapolating findings across different model systems will demand great care but promises to reveal a rich tapestry of themes in GRN architecture and regulation. The purpose of this review is to approach the field of leaf development from the perspectives of the evolution of developmental systems that orchestrate leaf development.

Long-distance transport of mRNA via parenchyma cells and phloem across the host-parasite junction in Cuscuta.

It has been shown that the parasitic plant dodder (Cuscuta pentagona) establishes a continuous vascular system through which water and nutrients are drawn. Along with solutes, viruses and proteins, mRNA transcripts are transported from the host to the parasite. The path of the transcripts and their stability in the parasite have yet to be revealed. To discover the route of mRNA transportation, the in situ reverse transcriptase-polymerase chain reaction (RT-PCR) technique was used to locally amplify host transcript within parasitic tissue. The stability of host mRNA molecules was also checked by monitoring specific transcripts along the growing dodder thread. Four mRNAs, alpha and beta subunits of PYROPHOSPHATE (PPi)-DEPENDENT PHOSPHOFRUCTOKINASE (LePFP), the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and GIBBERELLIC ACID INSENSITIVE (LeGAI), were found to move from host (tomato (Solanum lycopersicum)) to dodder. LePFP mRNA was localized to the dodder parenchyma cells and to the phloem. LePFP transcripts were found in the growing dodder stem up to 30 cm from the tomato-dodder connection. These results suggest that mRNA molecules are transferred from host to parasite via symplastic connections between parenchyma cells, move towards the phloem, and are stable for a long distance in the parasite. This may allow developmental coordination between the parasite and its host.

Compound leaf development and evolution in the legumes.

Across vascular plants, Class 1 KNOTTED1-like (KNOX1) genes appear to play a critical role in the development of compound leaves. An exception to this trend is found in the Fabaceae, where pea (Pisum sativum) uses UNIFOLIATA, an ortholog of the floral regulators FLORICAULA (FLO) and LEAFY (LFY), in place of KNOX1 genes to regulate compound leaf development. To assess the phylogenetic distribution of KNOX1-independent compound leaf development, a survey of KNOX1 protein expression across the Fabaceae was undertaken. The majority of compound-leafed Fabaceae have expression of KNOX1 proteins associated with developing compound leaves. However, in a large subclade of the Fabaceae, the inverted repeat-lacking clade (IRLC), of which pea is a member, KNOX1 expression is not associated with compound leaves. These data suggest that the FLO/LFY gene may function in place of KNOX1 genes in generating compound leaves throughout the IRLC. The contribution of FLO/LFY to leaf complexity in a member of the Fabaceae outside of the IRLC was examined by reducing expression of FLO/LFY orthologs in transgenic soybean (Glycine max). Transgenic plants with reduced FLO/LFY expression showed only slight reductions in leaflet number. Overexpression of a KNOX1 gene in alfalfa (Medicago sativa), a member of the IRLC, resulted in an increase in leaflet number. This implies that KNOX1 targets, which promote compound leaf development, are present in alfalfa and are still sensitive to KNOX1 regulation. These data suggest that KNOX1 genes and the FLO/LFY gene may have played partially overlapping roles in compound leaf development in ancestral Fabaceae but that the FLO/LFY gene took over this role in the IRLC.

Regulation of SHOOT MERISTEMLESS genes via an upstream-conserved noncoding sequence coordinates leaf development.

The indeterminate shoot apical meristem of plants is characterized by the expression of the Class 1 KNOTTED1-LIKE HOMEOBOX (KNOX1) genes. KNOX1 genes have been implicated in the acquisition and/or maintenance of meristematic fate. One of the earliest indicators of a switch in fate from indeterminate meristem to determinate leaf primordium is the down-regulation of KNOX1 genes orthologous to SHOOT MERISTEMLESS (STM) in Arabidopsis (hereafter called STM genes) in the initiating primordia. In simple leafed plants, this down-regulation persists during leaf formation. In compound leafed plants, however, KNOX1 gene expression is reestablished later in the developing primordia, creating an indeterminate environment for leaflet formation. Despite this knowledge, most aspects of how STM gene expression is regulated remain largely unknown. Here, we identify two evolutionarily conserved noncoding sequences within the 5' upstream region of STM genes in both simple and compound leafed species across monocots and dicots. We show that one of these elements is involved in the regulation of the persistent repression and/or the reestablishment of STM expression in the developing leaves but is not involved in the initial down-regulation in the initiating primordia. We also show evidence that this regulation is developmentally significant for leaf formation in the pathway involving ASYMMETRIC LEAVES1/2 (AS1/2) gene expression; these genes are known to function in leaf development. Together, these findings reveal a regulatory point of leaf development mediated through a conserved, noncoding sequence in STM genes.

Evolution of asexual reproduction in leaves of the genus Kalanchoë.

Plant somatic cells have the remarkable ability to regenerate an entire organism. Many species in the genus Kalanchoë, known as "mother of thousands," develop plantlets on the leaf margins. Using key regulators of organogenesis (STM) and embryogenesis (LEC1 and FUS3) processes, we analyzed asexual reproduction in Kalanchoë leaves. Suppression of STM abolished the ability to make plantlets. Here, we report that constitutive plantlet-forming species, like Kalanchoë daigremontiana, form plantlets by coopting both organogenesis and embryogenesis programs into leaves. These species have a defective LEC1 gene and produce nonviable seed, whereas species that produce plantlets only upon stress induction have an intact LEC1 gene and produce viable seed. The latter species are basal in the genus, suggesting that induced-plantlet formation and seed viability are ancestral traits. We provide evidence that asexual reproduction likely initiated as a process of organogenesis and then recruited an embryogenesis program into the leaves in response to loss of sexual reproduction within this genus.

L1 division and differentiation patterns influence shoot apical meristem maintenance.

Plant development requires regulation of both cell division and differentiation. The class 1 KNOTTED1-like homeobox (KNOX) genes such as knotted1 (kn1) in maize (Zea mays) and SHOOTMERISTEMLESS in Arabidopsis (Arabidopsis thaliana) play a role in maintaining shoot apical meristem indeterminacy, and their misexpression is sufficient to induce cell division and meristem formation. KNOX overexpression experiments have shown that these genes interact with the cytokinin, auxin, and gibberellin pathways. The L1 layer has been shown to be necessary for the maintenance of indeterminacy in the underlying meristem layers. This work explores the possibility that the L1 affects meristem function by disrupting hormone transport pathways. The semidominant Extra cell layers1 (Xcl1) mutation in maize leads to the production of multiple epidermal layers by overproduction of a normal gene product. Meristem size is reduced in mutant plants and more cells are incorporated into the incipient leaf primordium. Thus, Xcl1 may provide a link between L1 division patterns, hormonal pathways, and meristem maintenance. We used double mutants between Xcl1 and dominant KNOX mutants and showed that Xcl1 suppresses the Kn1 phenotype but has a synergistic interaction with gnarley1 and rough sheath1, possibly correlated with changes in gibberellin and auxin signaling. In addition, double mutants between Xcl1 and crinkly4 had defects in shoot meristem maintenance. Thus, proper L1 development is essential for meristem function, and XCL1 may act to coordinate hormonal effects with KNOX gene function at the shoot apex.