Morpho-Histology, Endogenous Hormone Dynamics and Transcriptome Profiling in Dacrydium Pectinatum during Male Cone Development

Dacrydium pectinatum de Laubenfels is a perennial gymnosperm species dominant in tropical montane rain forests. Due to severe damages by excessive deforestation, typhoons, and other external forces, the population of the species has been significantly reduced. Furthermore, its natural regeneration is poor. To better understand the male cone development in D. pectinatum, we examined the morphological and anatomical changes, analyzed the endogenous hormone dynamics, and profiled gene expression. The morpho-histological observations suggest that the development of D. pectinatum male cone can be largely divided into four stages: microspore primordium formation (April to May), microspore sac and pollen mother cell formation (July to November), pollen mother cell division (January), and pollen grain formation (February). The levels of gibberellins (GA), auxin (IAA), abscisic Acid (ABA), cytokinin (CTK), and jasmonic acid (JA) fluctuated during the process of male cone development. The first transcriptome database for a Dacrydium species was generated, revealing >70,000 unigene sequences. Differential expression analyses revealed several floral and hormone biosynthesis and signal transduction genes that could be critical for male cone development. Our study provides new insights on the cone development in D. pectinatum and the foundation for male cone induction with hormones and studies of factors contributing to the species’ low rate of seed germination.

Full-Length Transcriptome Analysis of Four Different Tissues of Cephalotaxus oliveri

Cephalotaxus oliveri is a tertiary relict conifer endemic to China, regarded as a national second-level protected plant in China. This species has experienced severe changes in temperature and precipitation in the past millions of years, adapting well to harsh environments. In view of global climate change and its endangered conditions, it is crucial to study how it responds to changes in temperature and precipitation for its conservation work. In this study, single-molecule real-time (SMRT) sequencing and Illumina RNA sequencing were combined to generate the complete transcriptome of C. oliveri. Using the RNA-seq data to correct the SMRT sequencing data, the four tissues obtained 63,831 (root), 58,108 (stem), 33,013 (leaf) and 62,436 (male cone) full-length unigenes, with a N50 length of 2523, 3480, 3181, and 3267 bp, respectively. Additionally, 35,887, 11,306, 36,422, and 25,439 SSRs were detected for the male cone, leaf, root, and stem, respectively. The number of long non-coding RNAs predicted from the root was the largest (11,113), and the other tissues were 3408 (stem), 3193 (leaf), and 3107 (male cone), respectively. Functional annotation and enrichment analysis of tissue-specific expressed genes revealed the special roles in response to environmental stress and adaptability in the different four tissues. We also characterized the gene families and pathways related to abiotic factors. This work provides a comprehensive transcriptome resource for C. oliveri, and this resource will facilitate further studies on the functional genomics and adaptive evolution of C. oliveri.

Temporal and Spatial Characteristics Endogenous Hormone Regulation During Male Cone Development of Dacrydium pectinatum

Background: Dacrydiumpectinatum de Laubenfels is a perennial gymnosperm dominant in tropical montane rain forests. Due to severe damages by excessive deforestation, typhoons, and other external forces, the population of the species has been significantly reduced. Furthermore, natural regeneration is poor. In order to better understand the reproductive process in D. pectinatum, we examined the morphological and anatomical changes during the development of male cone and analyzed the endogenous hormone dynamics.Results: Our study indicates that D. pectinatum male buds become distinguishable in April in tropical montane rain forests, while microspore sac forms in September and pollen mother cell forms and divides in December. Pollen grains mature and disperse in the following February. A mature male cone averages 8.5 mm in length. Level of GA, IAA, ABA and JA and their ratios fluctuated during late August to late November when sporogenous tissues were actively differentiated.Conclusions: The differentiation of sporogenous tissues is accompanied by variations in levels of endogenous hormones (GA, ABA, IAA, and JA) and their balances. The new insights about the cone development in D. pectinatum lay the foundation for future cone induction with hormones and study of factors contributing to the species’ low rate of seed germination.

Transcriptome-wide isolation and expression profiles of NF-Y gene family in male cone development and hormone treatment of Chinese pine (Pinus tabuliformis)

Background Conifers and angiosperms have difference in reproductive development, especially for flowering. It is known that NUCLEAR FACTOR Y (NF-Y) transcription factor play an important role in flowering, drought stress and GA, ABA signaling, but, little known in auxin, salicylic acid, jasmonic acid, etc. Moreover, the NF-Y genes family has been mainly analyzed in angiosperms, but it has not been comprehensively reported in conifers. Results In this study, we identified 9 NF-YA, 9 NF-YB, and 10 NF-YC genes in Pinus tabuliformis using Arabidopsis NF-Y protein sequences as queries. Besides, by comparing conserved regions and phylogenetic relationships of the PtNF-Ys, we found that the NF-Ys were both conserved and altered during evolution. PtTFL2, PtCO, PtNF-YC1 and PtNF-YC4 were exploited by expression profile in male cone development and the correlation analysis. In addition, NF-YC1/4 can interact with DPL by yeast two-hybrid assays and BiFC. The multiple types of phytohormones-responsive cis-elements (ABA, JA, IAA, SA) were present and many NF-Y genes responded positively to SA and as opposed to IAA and JA. Conclusions Twenty-eight PtNF-Ys were identified and bioinformatic characterization of NF-Y genes including conserved regions, phylogenetic relationships, gene-motifs, was carried out. Two candidate genes (NF-YC1 and NF-YC4) were found to be involved in the regulation of conifer flowering and gibberellin signalling. The cis-elements and hormone transcriptome analysis revealed that the potential role of NF-Ys in conifers resistance. This study provides the basis for improved understanding of NF-Y genes function in conifers.

Cycas micronesica Stem Carbohydrates Decline Following Leaf and Male Cone Growth Events

The growth of synchronized leaf flushes or male cones on Cycas trees is an ephemeral event, and non-structural carbohydrates (NSCs) are likely deployed from stem and root storage tissues to support their construction. The relationships among various stem NSCs and these rapid growth events have not been studied to date. Monosaccharides, disaccharides, and starch were quantified in Cycas micronesica stem tissue prior to and immediately after the growth of leaf flushes or male cones to determine the influences on the concentration of these carbohydrates. The pre-existing leaves were removed from half of the plants to determine if the elimination of this carbon source would influence the NSC behaviors. Starch and sucrose dominated the NSC profiles, and these two NSCs declined following cone or new leaf growth. Removal of pre-existing leaves generated a greater decline in starch and sucrose for cone growth, and a greater decline in sucrose, but not starch following new leaf growth than in control trees with no leaf removal. The initial differences in starch and sucrose among cortex, vascular, and pith tissues disappeared as the concentrations declined in all three tissue categories to reach similar post-growth concentrations among the stem tissue categories. The fructose, glucose, and maltose behaviors were not consistent, and their concentrations were low such that their influence on the total NSC behaviors was minimal. These results provided indirect evidence that stem NSCs were mobilized to support ephemeral male cone and new leaf growth for this arborescent cycad. Growth of female strobili is slow and lengthy, so we did not include female trees in this study. The contributions of stem NSCs to female strobili growth remain to be studied with alternative methods.

Supplementary description of Cycas hongheensis (Cycadaceae) from Yunnan, China and its phylogenetic position

Cycas hongheensis was firstly proposed as a distinct taxon in 1994 and formally described two years later based on vegetative materials only. Here, the reproductive organs of this species, namely, the female cone, male cone and seeds are supplemented for description as these fertile materials were found for the first time at the type locality. Furthermore, the phylogenetic position of this species as being the only endemic member of Cycas sect. Indosinenses in China was investigated. The conflicting phylogenic results between chloroplastic (cpDNA) and nuclear DNA (nDNA) are corresponded with the inconformity characters of morphology of this species. The characters of lacking indumentum on ovules and the distinct apical spine of megasporophyll are corresponding to nDNA result while the characters such as the soft and rudimentary apical spine of the microsporophyll and the absence of fibrous layer inside the sarcotesta comform to cpDNA result. For the taxonomy, we agree this species to be classified into Cycas sect. Indosinenses for the obvious morphological characters of megasporophyll and ovule as well as deeper evidence from phylogeny based on nuclear data, as biparental inherited nuclear genes could offer more comprehensive genetic information than maternal inherited cpDNA.