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

AbstractThe 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

A New Advanced Backcross Tomato Population Enables High Resolution Leaf QTL Mapping and Gene Identification.

Quantitative Trait Loci (QTL) mapping is a powerful technique for dissecting the genetic basis of traits and species differences. Established tomato mapping populations between domesticated tomato (Solanum lycopersicum) and its more distant interfertile relatives typically follow a near isogenic line (NIL) design, such as the S. pennellii Introgression Line (IL) population, with a single wild introgression per line in an otherwise domesticated genetic background. Here, we report on a new advanced backcross QTL mapping resource for tomato, derived from a cross between the M82 tomato cultivar and S. pennellii This so-called Backcrossed Inbred Line (BIL) population is comprised of a mix of BC2 and BC3 lines, with domesticated tomato as the recurrent parent. The BIL population is complementary to the existing S. pennellii IL population, with which it shares parents. Using the BILs, we mapped traits for leaf complexity, leaflet shape, and flowering time. We demonstrate the utility of the BILs for fine-mapping QTL, particularly QTL initially mapped in the ILs, by fine-mapping several QTL to single or few candidate genes. Moreover, we confirm the value of a backcrossed population with multiple introgressions per line, such as the BILs, for epistatic QTL mapping. Our work was further enabled by the development of our own statistical inference and visualization tools, namely a heterogeneous hidden Markov model for genotyping the lines, and by using state-of-the-art sparse regression techniques for QTL mapping

Long-Distance and Trans-Generational Stomatal Patterning by CO2 Across Arabidopsis Organs

Stomata control water loss and carbon dioxide uptake by both altering pore aperture and developmental patterning. Stomatal patterning is regulated by environmental factors including atmospheric carbon dioxide (p[CO2]), which is increasing globally at an unprecedented rate. Mature leaves are known to convey developmental cues to immature leaves in response to p[CO2], but the developmental mechanisms are unknown. To characterize changes in stomatal patterning resulting from signals moving from mature to developing leaves, we constructed a dual-chamber growth system in which rosette and cauline leaves of Arabidopsis thaliana were subjected to differing p[CO2]. Young rosette tissue was found to adjust stomatal index (SI, the proportion of stomata to total cell number) in response to both the current environment and the environment experienced by mature rosette tissue, whereas cauline leaves appear to be insensitive to p[CO2] treatment. It is likely that cauline leaves and cotyledons deploy mechanisms for controlling stomatal development that share common but also deploy distinctive mechanisms to that operating in rosette leaves. The effect of p[CO2] on stomatal development is retained in cotyledons of the next generation, however, this effect does not occur in pre-germination stomatal lineage cells but only after germination. Finally, these data suggest that p[CO2] affects regulation of stomatal development specifically through the development of satellite stomata (stomata induced by signals from a neighboring stomate) during spacing divisions and not the basal pathway. To our knowledge, this is the first report identifying developmental steps responsible for altered stomatal patterning to p[CO2] and its trans-generational inheritance

A High-Throughput Method for Illumina RNA-Seq Library Preparation.

With the introduction of cost effective, rapid, and superior quality next generation sequencing techniques, gene expression analysis has become viable for labs conducting small projects as well as large-scale gene expression analysis experiments. However, the available protocols for construction of RNA-sequencing (RNA-Seq) libraries are expensive and/or difficult to scale for high-throughput applications. Also, most protocols require isolated total RNA as a starting point. We provide a cost-effective RNA-Seq library synthesis protocol that is fast, starts with tissue, and is high-throughput from tissue to synthesized library. We have also designed and report a set of 96 unique barcodes for library adapters that are amenable to high-throughput sequencing by a large combination of multiplexing strategies. Our developed protocol has more power to detect differentially expressed genes when compared to the standard Illumina protocol, probably owing to less technical variation amongst replicates. We also address the problem of gene-length biases affecting differential gene expression calls and demonstrate that such biases can be efficiently minimized during mRNA isolation for library preparation

Regulation of Small RNA Accumulation in the Maize Shoot Apex

MicroRNAs (miRNAs) and trans-acting siRNAs (ta-siRNAs) are essential to the establishment of adaxial–abaxial (dorsoventral) leaf polarity. Tas3-derived ta-siRNAs define the adaxial side of the leaf by restricting the expression domain of miRNA miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants. To investigate the regulatory mechanisms that allow for the precise spatiotemporal accumulation of these polarizing small RNAs, we used laser-microdissection coupled to RT-PCR to determine the expression profiles of their precursor transcripts within the maize shoot apex. Our data reveal that the pattern of mature miR166 accumulation results, in part, from intricate transcriptional regulation of its precursor loci and that only a subset of mir166 family members contribute to the establishment of leaf polarity. We show that miR390, an upstream determinant in leaf polarity whose activity triggers tas3 ta-siRNA biogenesis, accumulates adaxially in leaves. The polar expression of miR390 is established and maintained independent of the ta-siRNA pathway. The comparison of small RNA localization data with the expression profiles of precursor transcripts suggests that miR166 and miR390 accumulation is also regulated at the level of biogenesis and/or stability. Furthermore, mir390 precursors accumulate exclusively within the epidermal layer of the incipient leaf, whereas mature miR390 accumulates in sub-epidermal layers as well. Regulation of miR390 biogenesis, stability, or even discrete trafficking of miR390 from the epidermis to underlying cell layers provide possible mechanisms that define the extent of miR390 accumulation within the incipient leaf, which patterns this small field of cells into adaxial and abaxial domains via the production of tas3-derived ta-siRNAs

Native Environment Modulates Leaf Size and Response to Simulated Foliar Shade across Wild Tomato Species

The laminae of leaves optimize photosynthetic rates by serving as a platform for both light capture and gas exchange, while minimizing water losses associated with thermoregulation and transpiration. Many have speculated that plants maximize photosynthetic output and minimize associated costs through leaf size, complexity, and shape, but a unifying theory linking the plethora of observed leaf forms with the environment remains elusive. Additionally, the leaf itself is a plastic structure, responsive to its surroundings, further complicating the relationship. Despite extensive knowledge of the genetic mechanisms underlying angiosperm leaf development, little is known about how phenotypic plasticity and selective pressures converge to create the diversity of leaf shapes and sizes across lineages. Here, we use wild tomato accessions, collected from locales with diverse levels of foliar shade, temperature, and precipitation, as a model to assay the extent of shade avoidance in leaf traits and the degree to which these leaf traits correlate with environmental factors. We find that leaf size is correlated with measures of foliar shade across the wild tomato species sampled and that leaf size and serration correlate in a species-dependent fashion with temperature and precipitation. We use far-red induced changes in leaf length as a proxy measure of the shade avoidance response, and find that shade avoidance in leaves negatively correlates with the level of foliar shade recorded at the point of origin of an accession. The direction and magnitude of these correlations varies across the leaf series, suggesting that heterochronic and/or ontogenic programs are a mechanism by which selective pressures can alter leaf size and form. This study highlights the value of wild tomato accessions for studies of both morphological and light-regulated development of compound leaves, and promises to be useful in the future identification of genes regulating potentially adaptive plastic leaf traits

Increases in Vein Length Compensate for Leaf Area Lost to Lobing in Grapevine

Premise:Leaf lobing and leaf size vary considerably across and within species,including among grapevines (Vitisspp.), some of the best‐studied leaves. Weexamined the relationship between leaf lobing and leaf area across grapevinepopulations that varied in extent of leaf lobing.Methods:We used homologous landmarking techniques to measure 2632 leavesacross 2 years in 476 unique, genetically distinct grapevines fromfive biparentalcrosses that vary primarily in the extent of lobing. We determined to what extent leafarea explained variation in lobing, vein length, and vein to blade ratio.Results:Although lobing was the primary source of variation in shape across theleaves we measured, leaf area varied only slightly as a function of lobing. Rather, leafarea increases as a function of total major vein length, total branching vein length, andvein to blade ratio. These relationships are stronger for more highly lobed leaves, withthe residuals for each model differing as a function of distal lobing.Conclusions:For leaves with different extents of lobing but the same area, the morehighly lobed leaves have longer veins and higher vein to blade ratios, allowing themto maintain similar leaf areas despite increased lobing. Thesefindings show howmore highly lobed leaves may compensate for what would otherwise result in areduced leaf area, allowing for increasedphotosynthetic capacity through similarleaf siz

Rootstock Effects on Scion Phenotypes in a ‘Chambourcin’ Experimental Vineyard

Understanding how root systems modulate shoot system phenotypes is a fundamental question in plant biology and will be useful in developing resilient agricultural crops. Grafting is a common horticultural practice that joins the roots (rootstock) of one plant to the shoot (scion) of another, providing an excellent method for investigating how these two organ systems affect each other. In this study, we used the French-American hybrid grapevine ‘Chambourcin’ (Vitis L.) as a model to explore the rootstock–scion relationship. We examined leaf shape, ion concentrations, and gene expression in ‘Chambourcin’ grown ungrafted as well as grafted to three different rootstocks (‘SO4’, ‘1103P’ and ‘3309C’) across 2 years and three different irrigation treatments. We found that a significant amount of the variation in leaf shape could be explained by the interaction between rootstock and irrigation. For ion concentrations, the primary source of variation identified was the position of a leaf in a shoot, although rootstock and rootstock by irrigation interaction also explained a significant amount of variation for most ions. Lastly, we found rootstock-specific patterns of gene expression in grafted plants when compared to ungrafted vines. Thus, our work reveals the subtle and complex effect of grafting on ‘Chambourcin’ leaf morphology, ionomics, and gene expression

Multi-dimensional Leaf Phenotypes Reflect Root System Genotype in Grafted Grapevine Over the Growing Season

Modern biological approaches generate volumes of multi-dimensional data, offering unprecedented opportunities to address biological questions previously beyond reach owing to small or subtle effects. A fundamental question in plant biology is the extent to which below-ground activity in the root system influences above-ground phenotypes expressed in the shoot system. Grafting, an ancient horticultural practice that fuses the root system of one individual (the rootstock) with the shoot system of a second, genetically distinct individual (the scion), is a powerful experimental system to understand below-ground effects on above-ground phenotypes. Previous studies on grafted grapevines have detected rootstock influence on scion phenotypes including physiology and berry chemistry. However, the extent of the rootstock\u27s influence on leaves, the photosynthetic engines of the vine, and how those effects change over the course of a growing season, are still largely unknown