Deciphering the evolution of the ovule genetic network through expression analyses in Gnetum gnemon.

BACKGROUND AND AIMS: The ovule is a synapomorphy of all seed plants (gymnosperms and angiosperms); however, there are some striking differences in ovules among the major seed plant lineages, such as the number of integuments or the orientation of the ovule. The genetics involved in ovule development have been well studied in the model species Arabidopsis thaliana, which has two integuments and anatropous orientation. This study is approached from what is known in arabidopsis, focusing on the expression patterns of homologues of four genes known to be key for the proper development of the integuments in arabidopsis: AINTEGUMENTA (ANT), BELL1, (BEL1), KANADIs (KANs) and UNICORN (UCN). METHODS: We used histology to describe the morphoanatomical development from ovules to seeds in Gnetum gnemon. We carried out spatiotemporal expression analyses in G. gnemon, a gymnosperm, which has a unique ovule morphology with an integument covering the nucellus, two additional envelopes where the outermost becomes fleshy as the seed matures, and an orthotropous orientation. KEY RESULTS: Our anatomical and developmental descriptions provide a framework for expression analyses in the ovule of G. gnemon. Our expression results show that although ANT, KAN and UCN homologues are expressed in the inner integument, their spatiotemporal patterns differ from those found in angiosperms. Furthermore, all homologues studied here are expressed in the nucellus, revealing major differences in seed plants. Finally, no expression of the studied homologues was detected in the outer envelopes. CONCLUSIONS: Altogether, these analyses provide significant comparative data that allows us to better understand the functional evolution of these gene lineages, providing a compelling framework for evolutionary and developmental studies of seeds. Our findings suggest that these genes were most likely recruited from the sporangium development network and became restricted to the integuments of angiosperm ovules.

Revealing the developmental dynamics in male strobilus transcriptome of Gnetum luofuense using nanopore sequencing technology.

Gnetum is a pantropical distributed gymnosperm genus. As being dioecious, Gnetum species apply female and male strobili to attract and provide nutrition to insect pollinators. Due to its unique gross morphology, a Gnetum male strobilus receives much attention in previous taxonomic and evolutionary studies. However, underlying molecular mechanisms that control male strobilus development and pollination adaptation have not been well studied. In the present study, nine full-length transcriptomes were sequenced from three developmental stages of the G. luofuense male strobili using Oxford Nanopore Technologies. In addition, weighted gene co-expression network analysis (WGCNA), and RT-qPCR analysis were performed. Our results show that a total of 3138 transcription factors and 466 long non-coding RNAs (lncRNAs) were identified, and differentially expressed lncRNAs and TFs reveal a dynamic pattern during the male strobilus development. Our results show that MADS-box and Aux/IAA TFs were differentially expressed at the three developmental stages, suggesting their important roles in the regulation of male strobilus development of G. luofuense. Results of WGCNA analysis and annotation of differentially expressed transcripts corroborate that the male strobilus development of G. luofuense is closely linked to plant hormone changes, photosynthesis, pollination drop secretion and reproductive organ defense. Our results provide a valuable resource for understanding the molecular mechanisms that drive organ evolution and pollination biology in Gnetum.

A full-length transcriptome and gene expression analysis reveal genes and molecular elements expressed during seed development in Gnetum luofuense.

BACKGROUND: Gnetum is an economically important tropical and subtropical gymnosperm genus with various dietary, industrial and medicinal uses. Many carbohydrates, proteins and fibers accumulate during the ripening of Gnetum seeds. However, the molecular mechanisms related to this process remain unknown. RESULTS: We therefore assembled a full-length transcriptome from immature and mature G. luofuense seeds using PacBio sequencing reads. We identified a total of 5726 novel genes, 9061 alternative splicing events, 3551 lncRNAs, 2160 transcription factors, and we found that 8512 genes possessed at least one poly(A) site. In addition, gene expression comparisons of six transcriptomes generated by Illumina sequencing showed that 14,323 genes were differentially expressed from an immature stage to a mature stage with 7891 genes upregulated and 6432 genes downregulated. The expression of 14 differentially expressed transcription factors from the MADS-box, Aux/IAA and bHLH families was validated by qRT-PCR, suggesting that they may have important roles in seed ripening of G. luofuense. CONCLUSIONS: These findings provide a valuable molecular resource for understanding seed development of gymnosperms.

Proteome-Level Analysis of Metabolism- and Stress-Related Proteins during Seed Dormancy and Germination in Gnetum parvifolium.

Gnetum parvifolium is a rich source of materials for traditional medicines, food, and oil, but little is known about the mechanism underlying its seed dormancy and germination. In this study, we analyzed the proteome-level changes in its seeds during germination using isobaric tags for relative and absolute quantitation. In total, 1,040 differentially expressed proteins were identified, and cluster analysis revealed the distinct time points during which signal transduction and oxidation-reduction activity changed. Gene Ontology analysis showed that "carbohydrate metabolic process" and "response to oxidative stress" were the main enriched terms. Proteins associated with starch degradation and antioxidant enzymes were important for dormancy-release, while proteins associated with energy metabolism and protein synthesis were up-regulated during germination. Moreover, protein-interaction networks were mainly associated with heat-shock proteins. Furthermore, in accord with changes in the energy metabolism- and antioxidant-related proteins, indole-3-acetic acid, Peroxidase, and soluble sugar content increased, and the starch content decreased in almost all six stages of dormancy and germination analyzed (S1-S6). The activity of superoxide dismutase, abscisic acid, and malondialdehyde content increased in the dormancy stages (S1-S3) and then decreased in the germination stages (S4-S6). Our results provide new insights into G. parvifolium seed dormancy and germination at the proteome and physiological levels, with implications for improving seed propagation.

Acclimation of seedlings of Gnetum leyboldii Tul. Gnetaceae to light changes in a tropical rain forest / Aclimatación de plántulas de Gnetum leyboldii Tul. Gnetaceae a los cambios de luz en un bosque lluvioso tropical

The neotropical liana Gnetum leyboldii Gnetaceae is a gymnosperm that resembles angiosperms in wood anatomy, overall morphology, and seed dispersal mechanism. Like other woody lianas, seedlings germinate in the shaded forest understory and start climbing towards the canopy, being eposed to sites with etreme differences in light conditions. However, the etent of physiological and structural adjustment to contrasting light conditions in the early regeneration stages of Gnetum is unknown. To answer this question, we analyzed seedling growth and photosynthetic responses using a common garden eperiment with two light regimes: full sun and low light 20 of full sun at La Selva Biological Station, Costa Rica. We also characterized the germination pattern of this species. We monitored one and half-month old seedlings for four months. Leaf structure finely adapted to light treatments, but gas echange properties were buffered by large seed reserves, which dominated biomass distribution about 50 of the total biomass, followed by stem 27, leaf 16 and root biomass 6 across light conditions. The presence of large seeds and the low photosynthetic rates of seedlings in both environments show that G. leyboldii is specialized to eploit deep shade. More research is needed to determine if the patterns found in G. leyboldii are typical of similar lianas that initially eploit deep-shaded understories in their ascension to the canopy.

La liana neotropical Gnetum leyboldii Gnetaceae es una gimnosperma que se asemeja a las angiospermas en la anatomía de la madera, morfología general de la planta y mecanismo de dispersión de semillas. Al igual que otras lianas leñosas, las plántulas se regeneran en el sotobosque bajo dosel cerrado y eventualmente ascienden hacia el dosel, eplotando sitios con diferencias etremas en condiciones lumínicas. Se desconoce el grado de ajuste fisiológico a condiciones lumínicas contrastantes en las primeras fases de regeneración de Gnetum. Para contestar esta pregunta, analizamos las respuestas de crecimiento de las plántulas a ambientes contrastantes de luz de sol y sombra en un jardín común con condiciones de alta cielo abierto y baja luminosidad 20 del ambiente de sol en la Estación Biológica La Selva, Costa Rica. También caracterizamos su patrón de germinación. Monitoreamos plántulas de 1.5 meses de edad por 4 meses. La estructura foliar mostró una fina adaptación a los tratamientos de luz, pero las propiedades de intercambio gaseoso no cambiaron sino que fueron amortiguadas por las reservas de las semillas grandes, las cuales dominaron la distribución de biomasa aproimadamente 50 de la biomasa total seguidas por el tallo 27, la hoja 16 y raíces 6. El tener semillas grandes y plántulas con bajas tasas fotosintéticas muestra que G. leyboldii en su etapa de plántula está adaptado para eplotar la sombra profunda. Se requiere más investigación para determinar si los patrones encontrados en G. leyboldii son típicos de otras lianas que inicialmente eplotan la sombra profunda en su ascensión al dosel.

Acclimation of seedlings of Gnetum leyboldii Tul. (Gnetaceae) to light changes in a tropical rain forest.

The neotropical liana Gnetum leyboldii (Gnetaceae) is a gymnosperm that resembles angiosperms in wood anatomy, overall morphology, and seed dispersal mechanism. Like other woody lianas, seedlings germinate in the shaded forest understory and start climbing towards the canopy, being exposed to sites with extreme differences in light conditions. However, the extent of physiological and structural adjustment to contrasting light conditions in the early regeneration stages of Gnetum is unknown. To answer this question, we analyzed seedling growth and photosynthetic responses using a common garden experiment with two light regimes: full sun and low light (20% of full sun) at La Selva Biological Station, Costa Rica. We also characterized the germination pattern of this species. We monitored one and half-month old seedlings for four months. Leaf structure finely adapted to light treatments, but gas exchange properties were buffered by large seed reserves, which dominated biomass distribution (about 50% of the total biomass), followed by stem (27%), leaf (16%) and root biomass (6%) across light conditions. The presence of large seeds and the low photosynthetic rates of seedlings in both environments show that G. leyboldii is specialized to exploit deep shade. More research is needed to determine if the patterns found in G. leyboldii are typical of similar lianas that initially exploit deep-shaded understories in their accession to the canopy.

Distinct MADS-box gene expression patterns in the reproductive cones of the gymnosperm Gnetum gnemon.

Expression patterns from in situ hybridization of four MADS-box genes (GGM7, GGM9, GGM11, and GGM15) from the gymnosperm species Gnetum gnemon are presented. Together with previously published data about putative orthologs of floral homeotic genes from G. gnemon (GGM2, GGM3, GGM13), we describe seven temporally and spatially distinct expression patterns in male, female or both types of reproductive units which very likely reflect the diversity of MADS-box gene function in gymnosperm cones. There is evidence that some aspects of the observed differential expression have been conserved since the last common ancestor of extant angiosperms and gymnosperms about 300 million years ago.