Multiplexed in situ hybridization reveals distinct lineage identities for major and minor vein initiation during maize leaf development.

Leaves of flowering plants are characterized by diverse venation patterns. Patterning begins with the selection of vein-forming procambial initial cells from within the ground meristem of a developing leaf, a process which is considered to be auxin-dependent, and continues until veins are anatomically differentiated with functional xylem and phloem. At present, the mechanisms responsible for leaf venation patterning are primarily characterized in the model eudicot Arabidopsis thaliana which displays a reticulate venation network. However, evidence suggests that vein development may proceed via a different mechanism in monocot leaves where venation patterning is parallel. Here, we employed Molecular Cartography, a multiplexed in situ hybridization technique, to analyze the spatiotemporal localization of a subset of auxin-related genes and candidate regulators of vein patterning in maize leaves. We show how different combinations of auxin influx and efflux transporters are recruited during leaf and vein specification and how major and minor vein ranks develop with distinct identities. The localization of the procambial marker PIN1a and the spatial arrangement of procambial initial cells that give rise to major and minor vein ranks further suggests that vein spacing is prepatterned across the medio-lateral leaf axis prior to accumulation of the PIN1a auxin transporter. In contrast, patterning in the adaxial-abaxial axis occurs progressively, with markers of xylem and phloem gradually becoming polarized as differentiation proceeds. Collectively, our data suggest that both lineage- and position-based mechanisms may underpin vein patterning in maize leaves.

Complementing model species with model clades.

Model species continue to underpin groundbreaking plant science research. At the same time, the phylogenetic resolution of the land plant Tree of Life continues to improve. The intersection of these two research paths creates a unique opportunity to further extend the usefulness of model species across larger taxonomic groups. Here we promote the utility of the Arabidopsis thaliana model species, especially the ability to connect its genetic and functional resources, to species across the entire Brassicales order. We focus on the utility of using genomics and phylogenomics to bridge the evolution and diversification of several traits across the Brassicales to the resources in Arabidopsis, thereby extending scope from a model species by establishing a "model clade". These Brassicales-wide traits are discussed in the context of both the model species Arabidopsis thaliana and the family Brassicaceae. We promote the utility of such a "model clade" and make suggestions for building global networks to support future studies in the model order Brassicales.

Two-tier morpho-chemical defence tactic in Aethionema via fruit morph plasticity and glucosinolates allocation in diaspores.

Fruit dimorphism and the production of glucosinolates (GSLs) are two specific life history traits found in the members of Brassicales, which aid to optimize seed dispersal and defence against antagonists, respectively. We hypothesized that the bipartite dispersal strategy demands a tight control over the production of fruit morphs with expectedly differential allocation of defensive anticipins (GSLs). In dimorphic Aethionema, herbivory by Plutella xylostella at a young stage triggered the production of more dehiscent (seeds released from fruit) than indehiscent fruit morphs (seeds enclosed within persistent pericarp) on the same plant upon maturity. Total GSL concentrations were highest in the mature seeds of dehiscent fruits from Aethionema arabicum and Aethionema saxatile among the different ontogenetic stages of the diaspores. Multivariate analyses of GSL profiles indicated significantly higher concentrations of specific indole GSLs in the diaspores, which require optimal defence after dispersal (i.e., seeds of dehiscent and fruit/pericarp of indehiscent fruit). Bioassays with a potentially coinhabitant fungus, Aspergillus quadrilineatus, support the distinct defensive potential of the diaspores corresponding to their GSL allocation. These findings indicate a two-tier morpho-chemical defence tactic of Aethionema via better protected fruit morphs and strategic provision of GSLs that optimize protection to the progeny for survival in nature.

Molecular phylogeny of 21 tropical bamboo species reconstructed by integrating non-coding internal transcribed spacer (ITS1 and 2) sequences and their consensus secondary structure.

The unavailability of the reproductive structure and unpredictability of vegetative characters for the identification and phylogenetic study of bamboo prompted the application of molecular techniques for greater resolution and consensus. We first employed internal transcribed spacer (ITS1, 5.8S rRNA and ITS2) sequences to construct the phylogenetic tree of 21 tropical bamboo species. While the sequence alone could grossly reconstruct the traditional phylogeny amongst the 21-tropical species studied, some anomalies were encountered that prompted a further refinement of the phylogenetic analyses. Therefore, we integrated the secondary structure of the ITS sequences to derive individual sequence-structure matrix to gain more resolution on the phylogenetic reconstruction. The results showed that ITS sequence-structure is the reliable alternative to the conventional phenotypic method for the identification of bamboo species. The best-fit topology obtained by the sequence-structure based phylogeny over the sole sequence based one underscores closer clustering of all the studied Bambusa species (Sub-tribe Bambusinae), while Melocanna baccifera, which belongs to Sub-Tribe Melocanneae, disjointedly clustered as an out-group within the consensus phylogenetic tree. In this study, we demonstrated the dependability of the combined (ITS sequence+structure-based) approach over the only sequence-based analysis for phylogenetic relationship assessment of bamboo.

WRKY6 restricts Piriformospora indica-stimulated and phosphate-induced root development in Arabidopsis.

BACKGROUND: Arabidopsis root growth is stimulated by Piriformospora indica, phosphate limitation and inactivation of the WRKY6 transcription factor. Combinations of these factors induce unexpected alterations in root and shoot growth, root architecture and root gene expression profiles. RESULTS: The results demonstrate that P. indica promotes phosphate uptake and root development under Pi limitation in wrky6 mutant. This is associated with the stimulation of PHOSPHATE1 expression and ethylene production. Expression profiles from the roots of wrky6 seedlings identified genes involved in hormone metabolism, transport, meristem, cell and plastid proliferation, and growth regulation. 25 miRNAs were also up-regulated in these roots. We generated and discuss here a list of common genes which are regulated in growing roots and which are common to all three growth stimuli investigated in this study. CONCLUSION: Since root development of wrky6 plants exposed to P. indica under phosphate limitation is strongly promoted, we propose that common genes which respond to all three growth stimuli are central for the control of root growth and architecture. They can be tested for optimizing root growth in model and agricultural plants.

The beneficial fungus Piriformospora indica protects Arabidopsis from Verticillium dahliae infection by downregulation plant defense responses.

BACKGROUND: Verticillium dahliae (Vd) is a soil-borne vascular pathogen which causes severe wilt symptoms in a wide range of plants. The microsclerotia produced by the pathogen survive in soil for more than 15 years. RESULTS: Here we demonstrate that an exudate preparation induces cytoplasmic calcium elevation in Arabidopsis roots, and the disease development requires the ethylene-activated transcription factor EIN3. Furthermore, the beneficial endophytic fungus Piriformospora indica (Pi) significantly reduced Vd-mediated disease development in Arabidopsis. Pi inhibited the growth of Vd in a dual culture on PDA agar plates and pretreatment of Arabidopsis roots with Pi protected plants from Vd infection. The Pi-pretreated plants grew better after Vd infection and the production of Vd microsclerotia was dramatically reduced, all without activating stress hormones and defense genes in the host. CONCLUSIONS: We conclude that Pi is an efficient biocontrol agent that protects Arabidopsis from Vd infection. Our data demonstrate that Vd growth is restricted in the presence of Pi and the additional signals from Pi must participate in the regulation of the immune response against Vd.

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.

Single-cell transcriptome atlases of soybean root and mature nodule reveal new regulatory programs that control the nodulation process.

The soybean root system is complex. In addition to being composed of various cell types, the soybean root system includes the primary root, the lateral roots, and the nodule, an organ in which mutualistic symbiosis with N-fixing rhizobia occurs. A mature soybean root nodule is characterized by a central infection zone where atmospheric nitrogen is fixed and assimilated by the symbiont, resulting from the close cooperation between the plant cell and the bacteria. To date, the transcriptome of individual cells isolated from developing soybean nodules has been established, but the transcriptomic signatures of cells from the mature soybean nodule have not yet been characterized. Using single-nucleus RNA-seq and Molecular Cartography technologies, we precisely characterized the transcriptomic signature of soybean root and mature nodule cell types and revealed the co-existence of different sub-populations of B. diazoefficiens-infected cells in the mature soybean nodule, including those actively involved in nitrogen fixation and those engaged in senescence. Mining of the single-cell-resolution nodule transcriptome atlas and the associated gene co-expression network confirmed the role of known nodulation-related genes and identified new genes that control the nodulation process. For instance, we functionally characterized the role of GmFWL3, a plasma membrane microdomain-associated protein that controls rhizobial infection. Our study reveals the unique cellular complexity of the mature soybean nodule and helps redefine the concept of cell types when considering the infection zone of the soybean nodule.

The post-pollination ethylene burst and the continuation of floral advertisement are harbingers of non-random mate selection in Nicotiana attenuata.

The self-compatible plant Nicotiana attenuata grows in genetically diverse populations after fires, and produces flowers that remain open for 3 days and are visited by assorted pollinators. To determine whether and when post-pollination non-random mate selection occurs among self and non-self pollen, seed paternity and semi-in vivo pollen tube growth were determined in controlled single/mixed pollinations. Despite all pollen sources being equally proficient in siring seeds in single-genotype pollinations, self pollen was consistently selected in mixed pollinations, irrespective of maternal genotype. However, clear patterns of mate discrimination occurred amongst non-self pollen when mixed pollinations were performed soon after corollas open, including selection against hygromycin B resistance (transformation selectable marker) in wild-type styles and for it in transformed styles. However, mate choice among pollen genotypes was completely shut down in plants transformed to be unable to produce (irACO) or perceive (ETR1) ethylene. The post-pollination ethylene burst, which originates primarily from the stigma and upper style, was strongly correlated with mate selection in single and mixed hand-pollinations using eight pollen donors in two maternal ecotypes. The post-pollination ethylene burst was also negatively correlated with the continuation of emission of benzylacetone, the most abundant pollinator-attracting corolla-derived floral volatile. We conclude that ethylene signaling plays a pivotal role in mate choice, and the post-pollination ethylene burst and the termination of benzylacetone release are accurate predictors, both qualitatively and quantitatively, of pre-zygotic mate selection and seed paternity.

Unpredictability of nectar nicotine promotes outcrossing by hummingbirds in Nicotiana attenuata.

Many plants use sophisticated strategies to maximize their reproductive success via outcrossing. Nicotiana attenuata flowers produce nectar with nicotine at concentrations that are repellent to hummingbirds, increasing the number of flowers visited per plant. In choice tests using native hummingbirds, we show that these important pollinators learn to tolerate high-nicotine nectar but prefer low-nicotine nectar, and show no signs of nicotine addiction. Nectar nicotine concentrations, unlike those of other vegetative tissues, are unpredictably variable among flowers, not only among populations, but also within populations, and even among flowers within an inflorescence. To evaluate whether variations in nectar nicotine concentrations increase outcrossing, polymorphic microsatellite markers, optimized to evaluate paternity in native N. attenuata populations, were used to compare outcrossing in plants silenced for expression of a biosynthetic gene for nicotine production (Napmt1/2) and in control empty vector plants, which were antherectomized and transplanted into native populations. When only exposed to hummingbird pollinators, seeds produced by flowers with nicotine in their nectar had a greater number of genetically different sires, compared to seeds from nicotine-free flowers. As the variation in nectar nicotine levels among flowers in an inflorescence decreased in N. attenuata plants silenced in various combinations of three Dicer-like (DCL) proteins, small RNAs are probably involved in the unpredictable variation in nectar nicotine levels within a plant.

The effect of dynamic twisting on the flow field and the unsteady forces of a heaving flat plate.

Many marine animals can dynamically twist their pectoral fins while swimming. The effects of such dynamic twisting on the unsteady forces on the fin and its surrounding flow field are yet to be understood in detail. In this paper, a flat plate executing a heaving maneuver is subjected to a similar dynamic twisting. In particular, the effects of the direction of twist, non-dimensional heaving amplitude, and reduced frequency are studied using a force sensor and particle image velocimetry (PIV) measurements. Two reduced frequencies,k=0.105, and0.209, and two twisting modes are investigated. In the first twisting mode, the plate is twisted in the direction of the heave (forward-twist), and in the second mode, the plate is twisted opposite to the direction of the heave (backward-twist). Force sensor measurements show that the forward-twist recovers some of the lift that is usually lost during the upstroke of flapping locomotion. Additionally, the forward-twist maintains a near-constant lift coefficient during the transition between downstroke and upstroke, suggesting a more stable form of locomotion. PIV results show that forward-twist limits circulation and leading-edge vortex growth during the downstroke, keepingCd≈0at the cost of the reduced lift. By contrast, backward-twist increases the circulation during the downstroke, resulting in large increases in both lift and drag coefficients. Force sensor data also showed that this effect on the lift is reversed during the upstroke, where the backward-twist causes a negative lift. The effects of each twisting mode are mainly caused by the changes in the shear layer velocity that occur as a result of twisting about the spanwise axis along the mid-chord. The twisting performed by forward-twist reduces the effective angle of attack through the upstroke and downstroke, resulting in a reduced shear layer velocity and lower circulation. The twisting performed by backward-twist does the exact opposite, increasing the effective angle of attack through the upstroke and downstroke and consequently increasing the shear layer velocity and circulation.

'Root of all success': Plasticity in root architecture of invasive wild radish for adaptive benefit.

Successful plant establishment in a particular environment depends on the root architecture of the seedlings and the extent of edaphic resource utilization. However, diverse habitats often pose a predicament on the suitability of the fundamental root structure of a species that evolved over a long period. We hypothesized that the plasticity in the genetically controlled root architecture in variable habitats provides an adaptive advantage to worldwide-distributed wild radish (Raphanus raphanistrum, Rr) over its close relative (R. pugioniformis, Rp) that remained endemic to the East Mediterranean region. To test the hypothesis, we performed a reciprocal comparative analysis between the two species, growing in a common garden experiment on their native soils (Hamra/Sandy for Rr, Terra Rossa for Rp) and complementary controlled experiments mimicking the major soil compositions. Additionally, we analyzed the root growth kinetics via semi-automated digital profiling and compared the architecture between Rr and Rp. In both experiments, the primary roots of Rr were significantly longer, developed fewer lateral roots, and showed slower growth kinetics than Rp. Multivariate analyses of seven significant root architecture variables revealed that Rr could successfully adapt to different surrogate growth conditions by only modulating their main root length and number of lateral roots. In contrast, Rp needs to modify several other root parameters, which are very resource-intensive, to grow on non-native soil. Altogether the findings suggest an evo-devo adaptive advantage for Rr as it can potentially establish in various habitats with the minimal tweak of key root parameters, hence allocating resources for other developmental requirements.

Morphologically and physiologically diverse fruits of two Lepidium species differ in allocation of glucosinolates into immature and mature seed and pericarp.

The morphology and physiology of diaspores play crucial roles in determining the fate of seeds in unpredictable habitats. In some genera of the Brassicaceae different types of diaspores can be found. Lepidium appelianum produces non-dormant seeds within indehiscent fruits while in L. campestre dormant seeds are released from dehiscent fruits. We investigated whether the allocation of relevant defence compounds into different tissues in different Lepidium species may be related to the diverse dispersal strategy (indehiscent and dehiscent) and seed physiology (non-dormant and dormant). Total glucosinolate concentration and composition were analysed in immature and mature seeds and pericarps of L. appelianum and L. campestre using high-performance liquid chromatography. Moreover, for comparison, transgenic RNAi L. campestre lines were used that produce indehiscent fruits due to silencing of LcINDEHISCENCE, the INDEHISCENCE ortholog of L. campestre. Total glucosinolate concentrations were lower in immature compared to mature seeds in all studied Lepidium species and transgenic lines. In contrast, indehiscent fruits of L. appelianum maintained their total glucosinolate concentration in mature pericarps compared to immature ones, while in dehiscent L. campestre and in indehiscent RNAi-LcIND L. campestre a significant decrease in total glucosinolate concentrations from immature to mature pericarps could be detected. Indole glucosinolates were detected in lower abundance than the other glucosinolate classes (aliphatic and aromatic). Relatively high concentrations of 4-methoxyindol-3-ylmethyl glucosinolate were found in mature seeds of L. appelianum compared to other tissues, while no indole glucosinolates were detected in mature diaspores of L. campestre. The diaspores of the latter species may rather depend on aliphatic and aromatic glucosinolates for long-term protection. The allocation patterns of glucosinolates correlate with the morpho-physiologically distinct fruits of L. appelianum and L. campestre and may be explained by the distinct dispersal strategies and the dormancy status of both species.

Naturally-primed life strategy plasticity of dimorphic Aethionema arabicum facilitates optimal habitat colonization.

Plasticity in plant dispersal traits can maximise the ability of a plant species to survive in stressful environments during colonization. Aethionema arabicum (Brassicaceae) is a dimorphic annual species that is hypothesized to survive stressful conditions during colonization due to adaptive plasticity in life-phase (vegetative vs sexual) and fruit morph (dehiscent [DEH] vs indehiscent fruits [IND]). We tested for adaptive plasticity in life-phase and fruit morphs along laboratory environmental stress gradients found in the natural habitats of Ae. arabicum. We considered optimal environmental conditions (750-2000 m above sea level) to be those that resulted in the following fitness parameters: higher biomass and a higher total number of fruits compared to stressful habitats. We found evidence of plasticity in life-phase and fruit-morph along a stressful environmental gradient. High hydrothermal stress proportionally increased the number of dehiscent morphs and non-dormant seeds germinating in autumn. This offsets natural phenology towards dry and cold winter (less hydrothermal stress), yielding fewer fruits that dehisce in the next generation. We conclude that the plastic responses of Ae. arabicum to natural stress gradients constitute a strategy of long-term adaptive benefits and favouring potential pathways of colonisation of the optimal habitat.

Morphological and molecular characterization of Bambusa tulda with a note on flowering.

BACKGROUND AND AIMS: Flowering incidence in Bambusa tulda has a high socio-economic impact. The aim of the present study was to describe the species for morphological characters (vegetative and reproductive) as well as molecular markers in order to enable species identification at various stages of the life cycle. METHODS: Thirty-two key morphological characters (15 culm and 17 culm-sheath) were studied along with detailed inflorescence and floral characters. Incidence of sporadic flowering was recorded. Genomic DNA was isolated from leaves collected from 17 eco-geographical locations and RAPD profiles were generated. KEY RESULTS: The description of culm, culm-sheath, inflorescence and floral morphology are in agreement with the prior taxonomic description by Gamble in 1896, but in this communication a more detailed description and illustrations are presented. No seed set was recorded following sporadic flowering, probably due to prezygoting isolating mechanisms (herkogamy or protandry). All 17 populations surveyed generated identical RAPD profiles. CONCLUSIONS: Sporadic flowering may occur in B. tulda, but may not necessarily be followed by gregarious flowering, and does not result in seed production.

Generation and characterization of SCARs by cloning and sequencing of RAPD products: a strategy for species-specific marker development in bamboo.

BACKGROUND AND AIMS: The aim of this study was to develop species-specific molecular markers for Bambusa balcooa and B. tulda to allow for their proper identification, in order to avoid unintentional adulteration that affects the quality and quantity of paper pulp production. METHODS: Two putative, species-specific RAPD markers, Bb836 for B. balcooa and Bt609 for B. tulda were generated using a PCR-based RAPD technique. Species-specificity of these two markers was confirmed through Southern hybridization in which RAPD gels were blotted and hybridized with radiolabelled cloned RAPD markers. Southern hybridization analyses were also performed to validate homology of the co-migrating Bb836 and Bt609 marker bands amplified from 16 different populations of B. balcooa and B. tulda, respectively. Sequence-characterized amplified region (SCAR) markers were developed from Bb836 and Bt609 sequences, using 20-mer oligonucleotide primers designed from both the flanking ends of the respective RAPD primers. KEY RESULTS: As anticipated, Bb836 hybridized with an amplified band from B. balcooa and Bt609 hybridized only with an amplified product from B. tulda; the two markers did not hybridize with the amplified products of any of the other 14 bamboo species studied. The two pairs of SCAR primers amplified the target sequences only in the respective species. The species-specific SCAR fragments were named as 'Balco836' for B. balcooa and 'Tuldo609' for B. tulda. The species-specific 'Balco836' was amplified from the genomic DNA of 80 individuals of 16 populations of B. balcooa studied. Similarly, the presence of 'Tuldo609' was noted in all the 80 individuals representing 16 populations of B. tulda assessed. These SCAR fragments contained no obvious repetitive sequence beyond the primers. CONCLUSION: These two molecular markers are potentially useful for regulatory agencies to establish sovereign rights of the germplasms of B. balcooa and B. tulda. In addition, this is the first report of species-specific SCAR marker development in bamboo.