Evolution and development of fruits of Erycina pusilla and other orchid species

Fruits play a crucial role in seed dispersal. They open along dehiscence zones. Fruit dehiscence zone formation has been intensively studied in Arabidopsis thaliana. However, little is known about the mechanisms and genes involved in the formation of fruit dehiscence zones in species outside the Brassicaceae. The dehiscence zone of A. thaliana contains a lignified layer, while dehiscence zone tissues of the emerging orchid model Erycina pusilla include a lipid layer. Here we present an analysis of evolution and development of fruit dehiscence zones in orchids. We performed ancestral state reconstructions across the five orchid subfamilies to study the evolution of selected fruit traits and explored dehiscence zone developmental genes using RNA-seq and qPCR. We found that erect dehiscent fruits with non-lignified dehiscence zones and a short ripening period are ancestral characters in orchids. Lignified dehiscence zones in orchid fruits evolved multiple times from non-lignified zones. Furthermore, we carried out gene expression analysis of tissues from different developmental stages of E. pusilla fruits. We found that fruit dehiscence genes from the MADS-box gene family and other important regulators in E. pusilla differed in their expression pattern from their homologs in A. thaliana. This suggests that the current A. thaliana fruit dehiscence model requires adjustment for orchids. Additionally, we discovered that homologs of A. thaliana genes involved in the development of carpel, gynoecium and ovules, and genes involved in lipid biosynthesis were expressed in the fruit valves of E. pusilla, implying that these genes may play a novel role in formation of dehiscence zone tissues in orchids. Future functional analysis of developmental regulators, lipid identification and quantification can shed more light on lipid-layer based dehiscence of orchid fruits

Orchid fruit and root movement analyzed using 2D photographs and a bioinformatics pipeline for processing sequential 3D scans

Abstract Premise Most studies of the movement of orchid fruits and roots during plant development have focused on morphological observations; however, further genetic analysis is required to understand the molecular mechanisms underlying this phenomenon. A precise tool is required to observe these movements and harvest tissue at the correct position and time for transcriptomics research. Methods We utilized three‐dimensional (3D) micro–computed tomography (CT) scans to capture the movement of fast‐growing Erycina pusilla roots, and built an integrated bioinformatics pipeline to process 3D images into 3D time‐lapse videos. To record the movement of slowly developing E. pusilla and Phalaenopsis equestris fruits, two‐dimensional (2D) photographs were used. Results The E. pusilla roots twisted and resupinated multiple times from early development. The first period occurred in the early developmental stage (77–84 days after germination [DAG]) and the subsequent period occurred later in development (140–154 DAG). While E. pusilla fruits twisted 45° from 56–63 days after pollination (DAP), the fruits of P. equestris only began to resupinate a week before dehiscence (133 DAP) and ended a week after dehiscence (161 DAP). Discussion Our methods revealed that each orchid root and fruit had an independent direction and degree of torsion from the initial to the final position. Our innovative approaches produced detailed spatial and temporal information on the resupination of roots and fruits during orchid development

<b>The 3D time-lapse video of young and mature plants of </b><b><i>E. pusilla</i></b><b>, produced using Avizo and bioinformatics pipeline.</b>

These time-lapse movies show the root resupination in young and mature plants of E. pusilla orchid plants. These movies are generated using the Avizo program and our bioinformatics pipeline. Below, you will find specific information about each of these videos:Movie 1. The 3D time-lapse video of a young plant of E. pusilla, produced using Avizo.Movie 2. The 3D time-lapse video of a mature plant of E. pusilla, produced using Avizo.Movie 3. The 3D time-lapse video of a young plant of E. pusilla, produced from a bioinformatics pipeline.Movie 4. The 3D time-lapse video of a mature plant of E. pusilla, produced from a bioinformatics pipeline.</p

<b>Supplementary information for the manuscript titled "Analysis of Fruit and Root Movement in Two Orchid Species Using 2D Photographs and a Bioinformatics Pipeline for Sequential 3D Scans Processing"</b>

The Supporting Information comprises 11 appendices, which include:Appendix S1. CT scan protocol of young, mature, early, and late flowering plants of Erycina pusilla.Appendix S2. 3D CT scan contrast test of different media in which Erycina pusilla was grown: (A) gelrite; B) glass beads; C) sand; D) solid agar; E) Sphagnum moss; and F) cotton). Scale bar: 1 cm.Appendix S3. Scanning and reconstructing images of Erycina pusilla plants with SkyScan1172 Micro CT: a step-by-step guide.Appendix S4. Response of five plants exposed to weekly X-ray radiation compared to four control plants not exposed to X-rays.Appendix S5. Effect of repeated exposure to X-rays during CT-scanning over 19 weeks on development of young, mature, early, and fully flowering plants of Erycina pusilla compared with control plants not exposed to any X-rays.Appendix S6. Development of Erycina pusilla from seed to flowering stage recorded in the tissue cultivation laboratory of Naturalis Biodiversity Center. A) Leaf-like protocorm visible 28 days after germination (DAG); B) First development of roots at 35 DAG; C-G) seedling development 42-70 DAG; H-I) Vegetative plant stages 77-84.DAG; J-K) inflorescence development and initiation of resupination ± 180 degrees of the flower 91-98 DAG; L-M) Elongation of inflorescence stalk 105-112 DAG; O) Floral bud emerging from bract 119 DAG; P-Q) 30 degrees bending of pedicel until (R-S) flower opens 126-133 DAG. Scale bar: 1 cm.Appendix S7. Resupination of roots of Erycina pusilla after different exposure times to x-rays. Abbreviations: Root S-1: root of seedling; Root NF1-1: root of the first replication non-flowering plant; Root NF2-1= root number one of second replication of non-flowering plant; Root NF2-2= root number two of second replication of non-flowering plant; Root NF2-3= root number three of second replication of non-flowering plants.Appendix S8. Cell number data of P. equestris fruits analyzed.Appendix S9. Area measurement data of P. equestris fruits analyzed.Appendix S10. Number of cells of each valve in eight developmental stages of Phalaenopsis equestris fruits grown in the tissue cultivation laboratory of Naturalis Biodiversity Center. The number of cells is shown on the vertical axis, and the fruit developmental stages (days after pollination-DAP) are displayed on the horizontal axis. Abbreviations: Lab= fertile valve derived from labellum; P1= fertile valve derived from the first petal; P2= fertile valve derived from the second petal; S1= sterile valve originated from first lateral sepal; S2= sterile valve originated from dorsal sepal, and S3= sterile valve originated from second lateral sepal. Error bars are indicated in black.Appendix S11. Main morphological changes of Phalaenopsis equestris fruits (N=3) observed during development. Abbreviations DAP= days after pollination.</p

<b>Datasets of 3D reconstruction images of young and mature </b><b><i>E. pusilla</i></b><b> plants.</b>

Data 1 comprises 3D reconstruction images of young plants of E. pusilla orchid obtained through scanning with a SkyScan machine over a span of one to 19 weeks.Data 2 comprises 3D reconstruction images of mature plants of E. pusilla orchid obtained through scanning with a SkyScan machine over a span of one to 19 weeks.</p

Orchid fruit and root movement analyzed using 2D photographs and a bioinformatics pipeline for processing sequential 3D scans

Premise: Most studies of the movement of orchid fruits and roots during plant development have focused on morphological observations; however, further genetic analysis is required to understand the molecular mechanisms underlying this phenomenon. A precise tool is required to observe these movements and harvest tissue at the correct position and time for transcriptomics research. Methods: We utilized three-dimensional (3D) micro–computed tomography (CT) scans to capture the movement of fast-growing Erycina pusilla roots, and built an integrated bioinformatics pipeline to process 3D images into 3D time-lapse videos. To record the movement of slowly developing E. pusilla and Phalaenopsis equestris fruits, two-dimensional (2D) photographs were used. Results: The E. pusilla roots twisted and resupinated multiple times from early development. The first period occurred in the early developmental stage (77–84 days after germination [DAG]) and the subsequent period occurred later in development (140–154 DAG). While E. pusilla fruits twisted 45° from 56–63 days after pollination (DAP), the fruits of P. equestris only began to resupinate a week before dehiscence (133 DAP) and ended a week after dehiscence (161 DAP). Discussion: Our methods revealed that each orchid root and fruit had an independent direction and degree of torsion from the initial to the final position. Our innovative approaches produced detailed spatial and temporal information on the resupination of roots and fruits during orchid development

<b>Supplementary information for the manuscript titled "</b><b>Orchid fruit and root movement analysed with 2D photographs and a bioinformatics pipeline for processing sequential 3D scans".</b>

The Supporting Information comprises 11 appendices, which include:Appendix S1. 3D CT scan contrast test of different media in which Erycina pusilla was grown: (A) gelrite; B) glass beads; C) sand; D) solid agar; E) Sphagnum moss; and F) cotton). Scale bar: 1 cm.Appendix S2. CT scan protocol of young, mature, early, and late flowering plants of Erycina pusilla.Appendix S3. Scanning and reconstructing images of Erycina pusilla plants with SkyScan1172 Micro CT: a step-by-step guide.Appendix S4. Response of five plants exposed to weekly X-ray radiation compared to four control plants not exposed to X-rays.Appendix S5. Effect of repeated exposure to X-rays during CT-scanning over 19 weeks on development of young, mature, early, and fully flowering plants of Erycina pusilla compared with control plants not exposed to any X-rays.Appendix S6. Development of Erycina pusilla from seed to flowering stage recorded in the tissue cultivation laboratory of Naturalis Biodiversity Center. A) Leaf-like protocorm visible 28 days after germination (DAG); B) First development of roots at 35 DAG; C-G) seedling development 42-70 DAG; H-I) Vegetative plant stages 77-84.DAG; J-K) inflorescence development and initiation of resupination ± 180 degrees of the flower 91-98 DAG; L-M) Elongation of inflorescence stalk 105-112 DAG; O) Floral bud emerging from bract 119 DAG; P-Q) 30 degrees bending of pedicel until (R-S) flower opens 126-133 DAG. Scale bar: 1 cm.Appendix S7. Resupination of roots of Erycina pusilla after different exposure times to x-rays. Abbreviations: Root S-1: root of seedling; Root NF1-1: root of the first replication non-flowering plant; Root NF2-1= root number one of second replication of non-flowering plant; Root NF2-2= root number two of second replication of non-flowering plant; Root NF2-3= root number three of second replication of non-flowering plants.Appendix S8. Cell number data of P. equestris fruits analysed.Appendix S9. Area measurement data of P. equestris fruits analysed.Appendix S10. Number of cells of each valve in eight developmental stages of Phalaenopsis equestris fruits grown in the tissue cultivation laboratory of Naturalis Biodiversity Center. The number of cells is shown on the vertical axis, and the fruit developmental stages (days after pollination-DAP) are displayed on the horizontal axis. Abbreviations: Lab= fertile valve derived from labellum; P1= fertile valve derived from the first petal; P2= fertile valve derived from the second petal; S1= sterile valve originated from first lateral sepal; S2= sterile valve originated from dorsal sepal, and S3= sterile valve originated from second lateral sepal. Error bars are indicated in black.Appendix S11. Main morphological changes of Phalaenopsis equestris fruits (N=3) observed during development. Abbreviations DAP= days after pollination.These supplementary information are parts of the manuscript titled "Fruit and root movement of two orchid species analysed with 2D photographs and a bioinformatics pipeline for processing sequential 3D scans".</p

<b>Datasets of reconstruction images of young and mature </b><b><i>E. pusilla</i></b><b> plants.</b>

Data 1 comprises 3D reconstruction images of young plants of E. pusilla orchid obtained through scanning with a SkyScan machine over a span of one to 19 weeks.Data 2 comprises 3D reconstruction images of mature plants of E. pusilla orchid obtained through scanning with a SkyScan machine over a span of one to 19 weeks.Both of these datasets are parts of the manuscript titled "Analysis of Fruit and Root Movement in Two Orchid Species Using 2D Photographs and a Bioinformatics Pipeline for Processing Sequential 3D Scans".</p

<b>The 3D time-lapse videos of young and mature plants of</b><b> </b><b><i>E. pusilla </i></b><b>orchid</b><b> were generated using AVIZO and a new bioinformatics pipeline.</b>

Movie 1. The 3D time-lapse video of a young plant of E. pusilla, produced using AVIZO.Movie 2. The 3D time-lapse video of a mature plant of E. pusilla, produced using AVIZO.Movie 3. The 3D time-lapse video of a young plant of E. pusilla, produced from a bioinformatics pipeline.Movie 4. The 3D time-lapse video of a mature plant of E. pusilla, produced from a bioinformatics pipeline.These movies are parts of the manuscript titled "Analysis of Fruit and Root Movement in Two Orchid Species Using 2D Photographs and a Bioinformatics Pipeline for Processing Sequential 3D Scans."</p