Time-course transcriptome analysis reveals regulation of Arabidopsis seed dormancy by the transcription factors WOX11/12.

The induction of seed dormancy and its release involve a finely regulated genetic program controlled by various environmental and developmental cues that are critical for plant survival and population expansion. Light plays a key role in seed dormancy and germination, but the molecular mechanisms underlying the control of dormancy are unclear. In the present study, high-resolution temporal RNA-seq in Arabidopsis identified WOX11 as encoding a hub transcription factor during the seed dormancy induction and release stages. This gene might have evolved from gymnosperms and expanded in angiosperms with highly conserved expression patterns in seeds. WOX11 and its homolog WOX12 were highly expressed from 2 d after pollination, and mRNA abundance was greatly increased during the seed dormancy induction and release stages. Further, we found that WOX11 plays a role in the regulation of seed dormancy downstream of phytochrome B (PHYB)-mediated red-light signaling during the induction stage, indicating that WOX11/12 are newly identified components of red-light signaling transduction. Taken together, our results suggest that WOX11/12-mediated PHYB signaling regulates seed dormancy in Arabidopsis, and provide insights into the developmental regulation and evolutionary adaptation of plants to changes in the light environment.

Anatomy and RNA-Seq reveal important gene pathways regulating sex differentiation in a functionally Androdioecious tree, Tapiscia sinensis.

BACKGROUND: Gametogenesis is a key step in the production of ovules or pollen in higher plants. The sex-determination aspects of gametogenesis have been well characterized in the model plant Arabidopsis. However, little is known about this process in androdioecious plants. Tapiscia sinensis Oliv. is a functionally androdioecious tree, with both male and hermaphroditic individuals. Hermaphroditic flowers (HFs) are female-fertile flowers that can produce functional pollen and set fruits. However, compared with male flowers (MFs), the pollen viability and number of pollen grains per flower are markedly reduced in HFs. MFs are female-sterile flowers that fail to set fruit and that eventually drop. RESULTS: Compared with HF, a notable cause of MF female sterility in T. sinensis is when the early gynoecium meristem is disrupted. During the early stage of HF development (stage 6), the ring meristem begins to form as a ridge around the center of the flower. At this stage, the internal fourth-whorl organ is stem-like rather than carpelloid in MF. A total of 52,945 unigenes were identified as transcribed in MF and HF. A number of differentially expressed genes (DEGs) and metabolic pathways were detected as involved in the development of the gynoecium, especially the ovule, carpel and style. At the early gynoecium development stage, DEGs were shown to function in the metabolic pathways regulating ethylene biosynthesis and signal transduction (upstream regulator), auxin, cytokinin transport and signalling, and sex determination (or flower meristem identity). CONCLUSIONS: Pathways for the female sterility model were initially proposed to shed light on the molecular mechanisms of gynoecium development at early stages in T. sinensis.

Male-linked gene TsRPL10a' in androdioecious tree Tapiscia sinensis: implications for sex differentiation by influencing gynoecium development.

The mechanism of sex differentiation in androdioecy is of great significance for illuminating the origin and evolution of dioecy. Tapiscia sinensis Oliv. is a functionally androdioecious species with both male and hermaphroditic individuals. Male flowers of T. sinensis lack the ovules of gynoecia compared with hermaphrodites. To identify sex simply and accurately, and further find the potential determinants of sex differentiation in T. sinensis, we found that TsRPL10a', a duplicate of TsRPL10a, was a male-linked gene. The promoter (5' untranslated region and the first intron) of TsRPL10a' can be used to accurately identify sex in T. sinensis. TsRPL10a is a ribosomal protein that is involved in gynoecium development, and sufficient ribosomal levels are necessary for female gametogenesis. The expression level of TsRPL10a was significantly downregulated in male flower primordia compared with hermaphrodites. The RNA fluorescence in situ hybridization (FISH) assay demonstrated that TsRPL10a was almost undetectable in male gynoecia at the gynoecial ridge stage, which was a key period of ovule formation by scanning electron microscope observation. In male flowers, although the promoter activity of TsRPL10a was significantly higher than TsRPL10a' verified by transgenic Arabidopsis thaliana, the transcriptional expression ratio of TsRPL10a was obviously lower than TsRPL10a' and reached its lowest at the gynoecial ridge stage, indicating the existence of a female suppressor. The promoter similarity of TsRPL10a and TsRPL10a' was only 45.29%; the genomic sequence similarity was 89.8%; four amino acids were altered in TsRPL10a'. The secondary structure of TsRPL10a' was different from TsRPL10a, and TsRPL10a' did not exhibit FISH and GUS expression in the gynoecium the way TsRPL10a did. From the perspective of RT-qPCR, its high expression level, followed by the low expression level of TsRPL10a in male flowers, indicates its antagonism function with TsRPL10a. The evolutionary analysis, subcellular localization and flower expression pattern suggested that TsRPL10a might be functionally conserved with AtRPL10aA, AtRPL10aB and AtRPL10aC in A. thaliana. Overall, we speculated that TsRPL10a and its duplicate TsRPL10a' might be involved in sex differentiation by influencing gynoecium development in T. sinensis.

The de novo genome assembly of Tapiscia sinensis and the transcriptomic and developmental bases of androdioecy.

Tapiscia sinensis (Tapisciaceae) possesses an unusual androdioecious breeding system that has attracted considerable interest from evolutionary biologists. Key aspects of T. sinensis biology, including its biogeography, genomics, and sex-linked genes, are unknown. Here, we report the first de novo assembly of the genome of T. sinensis. The genome size was 410 Mb, with 22,251 predicted genes. Based on whole-genome resequencing of 55 trees from 10 locations, an analysis of population genetic structure indicated that T. sinensis has fragmented into five lineages, with low intrapopulation genetic diversity and little gene flow among populations. By comparing whole-genome scans of male versus hermaphroditic pools, we identified 303 candidate sex-linked genes, 79 of which (25.9%) were located on scaffold 25. A 24-kb region was absent in hermaphroditic individuals, and five genes in that region, TsF-box4, TsF-box10, TsF-box13, TsSUT1, and TsSUT4, showed expression differences between mature male and hermaphroditic flowers. The results of this study shed light on the breeding system evolution and conservation genetics of the Tapisciaceae.

Abnormal tapetum development in hermaphrodites of an androdioecious tree, Tapiscia sinensis.

Tapiscia sinensis Oliv. (Tapisciaceae) has been proven to be a functional androdioecious species with both male and hermaphroditic individuals, and the pollen viability of males is far higher than that of hermaphrodites. To better understand the causes of the low pollen viability in hermaphroditic flowers, different stages of anther development were observed. We found that hermaphroditic flowers exhibit abnormal tapetum development, resulting in low pollen viability. To clarify the underlying molecular mechanism of abnormal tapetum development in hermaphrodites, quantitative real-time PCR analyses were performed. The results revealed that the expression levels of an important transcription factor for tapetum development and function, T. sinensis DYSFUNCTIONAL TAPETUM1 (TsDYT1), and its potential downstream regulatory genes T. sinensis DEFECTIVE in TAPETAL DEVELOPMENT and FUNCTION1 (TsTDF1), T. sinensis ABORTED MICROSPORE (TsAMS) and T. sinensis MALE STERILITY 1 (TsMS1) were all significantly downregulated in hermaphrodites compared with males at some key stages of anther development. The amino acid sequence similarity, expression pattern, gene structure and subcellular localization of these genes were analyzed, and the results indicated functional conservation between T. sinensis and homologues in Arabidopsis thaliana. Next, rapid amplification of cDNA end and thermal asymmetric interlaced PCR were employed to clone the full-length cDNA and promoter sequences of these genes, respectively. In addition, results of yeast two-hybrid analysis showed that TsDYT1 can form heterodimers with TsAMS, and yeast one-hybrid analysis demonstrated that TsDYT1 directly binds to the promoter regions of TsTDF1 and TsMS1. TsTDF1 can directly regulate expression of TsAMS, suggesting that a functionally conserved pathway exists between A. thaliana and T. sinensis to regulate tapetum development. In conclusion, the results suggest that abnormal expression of core transcription factors for tapetum development, including TsDYT1, TsTDF1, TsAMS and TsMS1, plays an important role in the abnormal development of the tapetum in T. sinensis hermaphrodites. Furthermore, a hermaphroditic tapetum with abnormal function causes the low pollen viability of hermaphroditic trees. Our results provide new insight into our understanding of the underlying mechanism of why pollen viability is much higher in males than hermaphrodites of the androdioecious tree T. sinensis.

The complete chloroplast genome of Cinnamomum camphora (L.) Presl., a unique economic plant to China.

Cinnamomum camphora (Lauraceae) Presl. is a unique economic plant to China. The complete chloroplast (cp) genome was sequenced and assembled by using Illumina paired-end reads data. The circular cp genome is 152,729 bp in size, including a pair of inverted repeat (IRs) regions of 20,074 bp, a large single copy (LSC) region of 93,688 bp and a small single copy (SSC) region of 18,893 bp. The genome contains 127 unique genes, including 83 protein-coding genes (PCGs), 36 transfer RNA genes (tRNAs), and 8 ribosomal RNA genes (rRNAs). Besides, 19 genes possess a single intron, while another three genes (ycf3, rps12, and clpP) have a couple of introns. The GC content of entire C. camphora cp genome, LSC, SSC, and IR regions are 39.2, 38.0, 33.9, and 44.4%, respectively. Phylogenetic analysis based on the concatenated coding sequences of cp PCGs showed that C. camphora and Cinnamomum verum are closely related with each other within the genus of cinnamomum.

Complete chloroplast genome of an endangered tree species, Toona ciliata (Sapindales: Meliaceae).

The whole cp genome of T. ciliata was 159,502 bp in length, containing a pair of inverted repeat (26,961 bp for each), a large single copy (87,199 bp) and a small single copy (18,381 bp) regions. The cp genome encoded 138 genes, including 89 protein-coding genes, 40 tRNA genes, 8 rRNA genes and 1 pseudogene. The nucleotide composition was asymmetric (30.7% A, 19.3% C, 18.6% G and 31.4% T) with an overall GC content of 37.9%. The maximum likelihood phylogenetic analysis based on 21 complete cp genome sequences showed that T. ciliata closely related to Azadirachta indica.

Calcium oxalate degradation is involved in aerenchyma formation in Typha angustifolia leaves.

Typha angustifolia L. (Typhaceae) is an emergent aquatic plant, and aerenchyma is formed through cell lysis in its leaves. The developing aerenchyma of T. angustifolia contains many CaOx crystals (raphides). Oxalate oxidase (OXO) (oxalate:oxygen oxidoreductase, EC1.2.3.4) can degrades calcium oxalate to carbon dioxide and hydrogen peroxide (H2O2). High level of H2O2 acts as a key inducer for different types of developmentally and environmentally programmed cell death (PCD) and can promote the formation of aerenchyma. Therefore, the objective of this study was to describe the relationship between aerenchyma formation and the degradation of CaOx crystals. Light and transmission electron microscopy (TEM) results showed that CaOx crystals occurred between PCD-susceptible cells in the early phase of aerenchyma formation, and those cells and CaOx crystals were degraded at aerenchyma maturation. Cytochemical localisation was used to detect H2O2, and H2O2 was found in crystal idioblasts. In addition, the oxalate content, H2O2 content and OXO activity were determined. The results showed that the concentration of oxalate was the highest in the third cavity formation stage and the H2O2 concentration was also highest at this stage. Meanwhile, the activity of OXO was also high in the third cavity formation stage. TpOXO was highly expressed during the CaOx crystal degradation period by quantitative real-time PCR analysis. These results show that the degradation of CaOx crystals is involved in the regulation of the PCD process of aerenchyma. This study will contribute to understanding the changes in CaOx crystals during the formation of aerenchyma in T. angustifolia.

The complete chloroplast genome of Zelkova schneideriana (Rosales: Ulmaceae), an Endangered species endemic to China.

Zelkova schneideriana Hand.-Mazz. (Ulmaceae) is an endangered species endemic to China. In this study, we reported its complete chloroplast (cp) genome based on Illumina pair-end sequencing. The whole genome was 158,999 bp long, consisting of a pair of inverted repeat (IR) regions of 26,427 bp, a large single copy (LSC) region of 87,397 bp and a small single copy (SSC) region of 18,748 bp. The cp genome contained 133 genes, including 88 protein-coding genes (80 PCG species), 37 tRNA genes (30 tRNA species), and eight rRNA genes (4 rRNA species). The overall G+C content of the whole genome was 35.6%, and the corresponding values of the LSC, SSC, and IR regions were 33.0, 28.3, and 42.4%, respectively. The maximum likelihood phylogenetic analysis of 25 selected chloroplast genomes demonstrated that Z. schneideriana was closely related to Ulmus macrocarpa and Ulmus pumila.

Characterization of the complete chloroplast genome of Lycium barbarum (Solanales: Solanaceae), a unique economic plant to China.

Lycium barbarum (Solanaceae) is a unique economic plant to China. The complete chloroplast (cp)genome was sequenced and assembled by using Illumina paired-end reads data. The circular cp genome is 155,656 bp in size, including a pair of inverted repeat (IR) regions of 25,451 bp, a large single copy (LSC) region of 86,554 bp and a small single copy (SSC) region of 18,200 bp. Besides, 15 genes possess a single intron, while another three genes (clpP, rps12 and ycf3) have a couple of introns. The GC content of entire L. barbarum cp genome, LSC, SSC and IR regions are 37.8%, 35.9%, 32.3%, and 43.2%, respectively. Phylogenetic analysis based on the concatenated coding sequences of cp PCGs showed that L. barbarum and Atropa belladonna are closely related with each other within the family Solanaceae.

De novo transcriptomic analysis to identify differentially expressed genes during the process of aerenchyma formation in Typha angustifolia leaves.

Lysigenous aerenchyma is formed through programmed cell death (PCD) in Typha angustifolia leaves. However, the genome and transcriptome data for this species are unknown. To further elucidate the molecular basis of PCD during aerenchyma formation in T. angustifolia leaves, transcriptomic analysis of T. angustifolia leaves was performed using Illumina sequencing technology, revealing 73,821 unigenes that were produced by assembly of the reads in T1, T2 and T3 samples. The important pathways, such as programmed cell death (PCD), aerenchyma formation, and ethylene responsiveness were regulated by these unigenes. 1-aminocyclopropane-1-carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxidase (ACO) were highly up-regulated as key enzymes for ethylene synthesis, along with respiratory burst oxidase homolog (RBOH), metallothionein, calmodulin-like protein (CML), and polygalacturonase (PG), may collectively explain the PCD involved in T. angustifolia aerenchyma formation. We hypothesize that fermentation, metabolism and glycolysis generate ATP for PCD. We searched the 73,821 unigenes against protein databases, and 24,712 were annotated. Based on sequence homology, 16,012 of the 73,821 annotated unigenes were assigned to one or more Gene Ontology (GO) terms. Meanwhile, a total of 9537 unigenes were assigned to 126 pathways in the KEGG database. In summary, this investigation provides important guidelines for exploring the molecular mechanisms of aerenchyma formation in aquatic plants.