Summary Enabling data reuse and knowledge discovery is increasingly critical in modern science, and requires an effort towards standardising data publication practices. This is particularly challenging in the plant phenotyping domain, due to its complexity and heterogeneity. We have produced the MIAPPE 1.1 release, which enhances the existing MIAPPE standard in coverage, to support perennial plants, in structure, through an explicit data model, and in clarity, through definitions and examples. We evaluated MIAPPE 1.1 by using it to express several heterogeneous phenotyping experiments in a range of different formats, to demonstrate its applicability and the interoperability between the various implementations. Furthermore, the extended coverage is demonstrated by the fact that one of the datasets could not have been described under MIAPPE 1.0. MIAPPE 1.1 marks a major step towards enabling plant phenotyping data reusability, thanks to its extended coverage, and especially the formalisation of its data model, which facilitates its implementation in different formats. Community feedback has been critical to this development, and will be a key part of ensuring adoption of the standard.

Motivation Modern genomic breeding methods rely heavily on very large amounts of phenotyping and genotyping data, presenting new challenges in effective data management and integration. Recently, the size and complexity of datasets have increased significantly, with the result that data are often stored on multiple systems. As analyses of interest increasingly require aggregation of datasets from diverse sources, data exchange between disparate systems becomes a challenge. Results To facilitate interoperability among breeding applications, we present the public plant Breeding Application Programming Interface (BrAPI). BrAPI is a standardized web service API specification. The development of BrAPI is a collaborative, community-based initiative involving a growing global community of over a hundred participants representing several dozen institutions and companies. Development of such a standard is recognized as critical to a number of important large breeding system initiatives as a foundational technology. The focus of the first version of the API is on providing services for connecting systems and retrieving basic breeding data including germplasm, study, observation, and marker data. A number of BrAPI-enabled applications, termed BrAPPs, have been written, that take advantage of the emerging support of BrAPI by many databases. Availability and implementation More information on BrAPI, including links to the specification, test suites, BrAPPs, and sample implementations is available at https://brapi.org/. The BrAPI specification and the developer tools are provided as free and open source.

Cork cambium (or phellogen) is a secondary meristem responsible for the formation of phelloderm and phellem/cork, which together compose the periderm. In Quercus suber L., the phellogen is active throughout the entire life of the tree, producing a continuous and renewable outer bark of cork. To identify specific candidate genes associated with cork cambium activity and phellem differentiation, we performed a comparative transcriptomic study of Q. suber secondary growth tissues (xylem and phellogen/phellem) using RNA-seq. The present work provides a high-resolution map of all the transcripts identified in the phellogen/phellem tissues. A total of 6013 differentially expressed genes were identified, with 2875 of the transcripts being specifically enriched during the cork formation process versus secondary xylem formation. Furthermore, cork samples originating from the original phellogen (`virgin’ cork) and from a traumatic phellogen (`amadia’ cork) were also compared. Our results point to a shortlist of potentially relevant candidate genes regulating phellogen activity and phellem differentiation, including novel genes involved in the suberization process, as well as genes associated to ethylene and jasmonate signaling and to meristem function. The future functional characterization of some of the identified candidate genes will help to elucidate the molecular mechanisms underlying cork cambium activity and phellem differentiation.

Regulation of seed development by small non-coding RNAs (sRNAs) is an important mechanism controlling a crucial phase of the life cycle of seed plants. In this work, sRNAs from seed tissues (zygotic embryos and megagametophytes) and from somatic embryos of Pinus pinaster were analysed to identify putative regulators of seed/embryo development in conifers. In total, sixteen sRNA libraries covering several developmental stages were sequenced. We show that embryos and megagametophytes express a large population of 21-nt sRNAs and that substantial amounts of 24-nt sRNAs were also detected, especially in somatic embryos. A total of 215 conserved miRNAs, one third of which are conifer-specific, and 212 high-confidence novel miRNAs were annotated. MIR159, MIR171 and MIR394 families were found in embryos, but were greatly reduced in megagametophytes. Other families, like MIR397 and MIR408, predominated in somatic embryos and megagametophytes, suggesting their expression in somatic embryos is associated with in vitro conditions. Analysis of the predicted miRNA targets suggests that miRNA functions are relevant in several processes including transporter activity at the cotyledon-forming stage, and sulfur metabolism across several developmental stages. An important resource for studying conifer embryogenesis is made available here, which may also provide insightful clues for improving clonal propagation via somatic embryogenesis.

An increasing number of microRNAs (miRNAs) and miRNA‐related sequences produced during miRNA biogenesis, comprising the isomiRome, have been recently highlighted in different species as critical mediators of environmental stress responses. Conifers have some of the largest known genomes but an extensive characterization of the isomiRome from any conifer species has been lacking. We provide here a comprehensive overview of the Pinus pinaster isomiRome expressed in roots, stem and needles under well‐watered and drought conditions. From the 13,441 unique small RNA sequences identified, 2,980 were annotated as canonical miRNAs or miRNA* and the remaining were classified as isomiRNA or miRNA‐like sequences. A survey of their expression patterns highlighted roots as the most responsive organ under drought, where specific sequences of which a 24‐nt novel miRNA stood out, were strongly down‐regulated. Given the putative roles of the miRNA‐targeted transcripts validated specifically in root tissues, some of the miRNAs, conserved and novel, are shortlisted as potential regulators of drought response. These results provide a valuable resource for comparative studies between gymnosperms and angiosperms. Furthermore, it evidences high transferability of the isomiRome between pine species being a useful basis for further molecular regulation and physiological studies, and especially those focused on adaptation to drought conditions.

miRPursuit is a pipeline developed for running end-to-end analyses of high-throughput small RNA (sRNA) sequence data in model and nonmodel plants, from raw data to identified and annotated conserved and novel sequences. It consists of a series of UNIX shell scripts, which connect open-source sRNA analysis software. The involved parameters can be combined with convenient workflow management by users without advanced computational skills. miRPursuit presents several advantages when compared to other tools, including the possibility of processing several sRNA libraries in parallel, thus easily allowing a comparison of the differences in sRNA read accumulation among sRNA libraries. We validate miRPursuit by using datasets from a model plant and discuss its performance with the analysis of sRNAs from non-model species.

MicroRNAs (miRNAs) are key regulators of several plant developmental processes including embryogenesis. Most miRNA families are conserved across major groups of plant species, but their regulatory roles have been studied mainly in model species like Arabidopsis and other angiosperms. In gymnosperms, miRNA-dependent regulation has been less studied since functional approaches in these species are often difficult to establish. Given the fundamental roles of auxin signaling in somatic embryogenesis (SE) induction and embryo development, we investigated a previously predicted interaction between miR160 and a putative target encoding AUXIN RESPONSE FACTOR 18 in Pinus pinaster (PpARF18) embryonic tissues. Phylogenetic analysis of AUXIN RESPONSE FACTOR 18 (ARF18) from Pinus pinaster and Picea abies, used here as a model system of conifer embryogenesis, showed their close relatedness to AUXIN RESPONSE FACTOR (ARF) genes known to be targeted by miR160 in other species, including Arabidopsis ARF10 and ARF16. By using a luciferase (LUC) reporter system for miRNA activity in Arabidopsis protoplasts, we have confirmed that P. pinaster miR160 (ppi-miR160) interacts in vivo with PpARF18 target site. When the primary miR160 from P. pinaster was overexpressed in protoplasts under non-limiting levels of ARGONAUTE1, a significant increase of miR160 target cleavage activity was observed. In contrast, co-expression of the primary miRNA and the target mimic MIM160 led to a decrease of miR160 activity. Our results further support that this interaction is functional during consecutive stages of SE in the conifer model P. abies. Expression analyses conducted in five stages of development, from proembryogenic masses (PEMs) to the mature embryo, show that conifer ARF18 is negatively regulated by miR160 toward the fully developed mature embryo when miR160 reached its highest expression level. This study reports the first in vivo validation of a predicted target site of a conifer miRNA supporting the conservation of miR160 interaction with ARF targets in gymnosperms. The approach used here should be useful for future characterization of miRNA functions in conifer embryogenesis.

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