Renhua Zheng

Transcriptome Characteristics and Six Alternative Expressed Genes Positively Correlated with the Phase Transition of Annual Cambial Activities in Chinese Fir (Cunninghamia lanceolata (Lamb.) Hook)

Background The molecular mechanisms that govern cambial activity in angiosperms are well established, but little is known about these molecular mechanisms in gymnosperms. Chinese fir (Cunninghamia lanceolata (Lamb.) Hook), a diploid (2n  = 2x  = 22) gymnosperm, is one of the most important industrial and commercial timber species in China. Here, we performed transcriptome sequencing to identify the repertoire of genes expressed in cambium tissue of Chinese fir. Methodology/Principal Findings Based on previous studies, the four stage-specific cambial tissues of Chinese fir were defined using transmission electron microscopy (TEM). In total, 20 million sequencing reads (3.6 Gb) were obtained using Illumina sequencing from Chinese fir cambium tissue collected at active growth stage, with a mean length of 131 bp and a N50 of 90 bp. SOAPdenovo software was used to assemble 62,895 unigenes. These unigenes were further functionally annotated by comparing their sequences to public protein databases. Expression analysis revealed that the altered expression of six homologous genes (ClWOX1, ClWOX4, ClCLV1-like, ClCLV-like, ClCLE12, and ClPIN1-like) correlated positively with changes in cambial activities; moreover, these six genes might be directly involved in cambial function in Chinese fir. Further, the full-length cDNAs and DNAs for ClWOX1 and ClWOX4 were cloned and analyzed. Conclusions In this study, a large number of tissue/stage-specific unigene sequences were generated from the active growth stage of Chinese fir cambium. Transcriptome sequencing of Chinese fir not only provides extensive genetic resources for understanding the molecular mechanisms underlying cambial activities in Chinese fir, but also is expected to be an important foundation for future genetic studies of Chinese fir. This study indicates that ClWOX1 and ClWOX4 could be possible reverse genetic target genes for revealing the molecular mechanisms of cambial activities in Chinese fir.

The Complete Chloroplast Genome Sequence of a Relict Conifer Glyptostrobus pensilis: Comparative Analysis and Insights into Dynamics of Chloroplast Genome Rearrangement in Cupressophytes and Pinaceae.

Glyptostrobus pensilis, belonging to the monotypic genus Glyptostrobus (Family: Cupressaceae), is an ancient conifer that is naturally distributed in low-lying wet areas. Here, we report the complete chloroplast (cp) genome sequence (132,239 bp) of G. pensilis. The G. pensilis cp genome is similar in gene content, organization and genome structure to the sequenced cp genomes from other cupressophytes, especially with respect to the loss of the inverted repeat region A (IRA). Through phylogenetic analysis, we demonstrated that the genus Glyptostrobus is closely related to the genus Cryptomeria, supporting previous findings based on physiological characteristics. Since IRs play an important role in stabilize cp genome and conifer cp genomes lost different IR regions after splitting in two clades (cupressophytes and Pinaceae), we performed cp genome rearrangement analysis and found more extensive cp genome rearrangements among the species of cupressophytes relative to Pinaceae. Additional repeat analysis indicated that cupressophytes cp genomes contained less potential functional repeats, especially in Cupressaceae, compared with Pinaceae. These results suggested that dynamics of cp genome rearrangement in conifers differed since the two clades, Pinaceae and cupressophytes, lost IR copies independently and developed different repeats to complement the residual IRs. In addition, we identified 170 perfect simple sequence repeats that will be useful in future research focusing on the evolution of genetic diversity and conservation of genetic variation for this endangered species in the wild.

Spatial analysis increases efficiency of progeny testing of Chinese fir

We used spatial, global trend and post-blocking analysis to examine the effectiveness of a progeny trial in a tree breeding program for Chinese fir (Cunninghamia lanceolata (Lamb.) Hook) on a hilly site with an environmental gradient from hill top to bottom. Diameter at breast height (DBH) and tree height data had significant spatial auto-correlations among rows and columns. Adding a first-order separable autoregressive term more effectively modelled the spatial variation than did the incomplete block (IB) model used for the experimental design. The spatial model also accounted for effects of experimental design factors and greatly reduced residual variances. The spatial analysis relative to the IB analysis improved estimation of genetic parameters with the residual variance reduced 13 and 19% for DBH and tree height, respectively; heritability increased 35 and 51% for DBH and tree height, respectively; and genetic gain improved 3–5%. Fitting global trend and post-blocking did not improve the analyses under IB model. The use of a spatial model or combined with a design model is recommended for forest genetic trials, particularly with global trend and local spatial variation of hilly sites.

ClRTL1 Encodes a Chinese Fir RNase III–Like Protein Involved in Regulating Shoot Branching

Identification of genes controlling shoot branching is crucial for improving plant architecture and increasing crop yield or biomass. A branching mutant of Chinese fir named “Dugansha” (Cunninghamia lanceolata var. dugan.) has been isolated in our laboratory. We chose the cDNA-AFLP technique and an effective strategy to screen genes that potentially regulate shoot branching in Chinese fir using this mutant. An RNase III-like1 cDNA fragment named ClRTL1 was identified as a potential positive regulator. To investigate the function of ClRTL1 in regulating shoot branching, we cloned the full-length cDNA sequence from C. lanceolata (Lamb.) Hook, deduced its secondary structure and function, and overexpressed the coding sequence in Arabidopsis. The ClRTL1 cDNA is 1045 bp and comprises an open reading frame of 705 bp. It encodes a protein of 235 amino acids. The deduced secondary structure of the ClRTL1 indicates that it is a mini-RNase III-like protein. The expression analysis and phenotypes of 35S: ClRTL1 in A. thaliana implies that ClRTL1 plays a role in promoting shoot branching in Chinese fir.

Polysaccharides and lipids in the developing anthers of Hong Kong orchid tree (Bauhinia blakeana)

Key messageMicrospore ofBauhinia blakeanabegins to accumulate lipids, and starches appear following bicellular pollen development. The development of tapetal cells ofB. blakeanais unusual in different stage.AbstractThe distribution of starches and lipids in the developing anthers of Bauhinia blakeana was investigated using cytochemical methods. In young anthers, sporogenous cells accumulate some small lipids which remain in microspore mother cells but disappear during tetrad microspore development. Later in development, the microspore again begins to accumulate lipids, and some starches also appear following bicellular pollen development. At anthesis the nearly mature pollen grains accumulate abundant lipids and starches as fuel for subsequent pollen tube growth. Tapetal cells of B. blakeana are highly vacuolated at sporogenous stage and highly polar at tetrad stage. During the microspore stage, the large vacuoles of the tapetal cells decompose, the cell size decreases, and cell polarity disappears, leading to the formation of a layer of small cells containing density cytoplasm in which many lipids are synthesized, suggesting that in these cells, sugars are transformed into lipids to provide nutrition for the pollen. The structure and morphology of B. blakeana tapetal cells vary at different developmental stages, presenting a spatial–temporal pattern that appears to be coordinated with the process of pollen development.

Desiccation Treatment and Endogenous IAA Levels Are Key Factors Influencing High Frequency Somatic Embryogenesis in Cunninghamia lanceolata (Lamb.) Hook

Cunninghamia lanceolata (Lamb.) Hook (Chinese fir) is an important tree, commercially and ecologically, in southern China. The traditional regenerating methods are based on organogenesis and cutting propagation. Here, we report the development of a high-frequency somatic embryogenesis (SE) regeneration system synchronized via a liquid culture from immature zygotic embryos. Following synchronization, PEM II cell aggregates were developmentally equivalent in appearance to cleaved zygotic embryos. Embryo and suspensor growth and subsequent occurrence of the apical and then the cotyledonary meristems were similar for zygotic and SE embryo development. However, SE proembryos exhibited a more reddish coloration than zygotic proembryos, and SE embryos were smaller than zygotic embryos. Mature somatic embryos gave rise to plantlets on hormone-free medium. For juvenile explants, low concentrations of endogenous indole-3-acetic acid in initial explants correlated with improved proembryogenic mass formation, and high SE competency. Analysis of karyotypes and microsatellites detected no major genetic variation in the plants regenerated via SE, and suggest a potential in the further development of this system as a reliable methodology for true-to-type seedling production. Treatment with polyethylene glycol (PEG) and abscisic acid (ABA) were of great importance to proembryo formation and complemented each other. ABA assisted the growth of embryonal masses, whereas PEG facilitated the organization of the proembryo-like structures. SOMATIC EMBRYOGENESIS RECEPTOR KINASE SERK) and the WUSCHEL homeobox (WOX) transcription factor served as molecular markers during early embryogenesis. Our results show that ClSERKs are conserved and redundantly expressed during SE. SERK and WOX transcript levels were highest during development of the proembryos and lowest in developed embryos. ClWOX13 expression correlates with the critical transition from proembryogenic masses to proembryos. Both SERK and WOX expression reveal their applicability in Chinese fir as markers of early embryogenesis. Overall, the findings provided evidence for the potential of this system in high fidelity Chinese fir seedlings production. Also, SE modification strategies were demonstrated and could be applied in other conifer species on the basis of our hormonal, morphological and molecular analyses.