Xinlei Li

Genome-wide transcriptome profiling provides insights into floral bud development of summer-flowering Camellia azalea

The transition from vegetative to reproductive growth in woody perennials involves pathways controlling flowering timing, bud dormancy and outgrowth in responses to seasonal cues. However little is known about the mechanism governing the adaptation of signaling pathways to environmental conditions in trees. Camellia azalea is a rare species in this genus flowering during summer, which provides a unique resource for floral timing breeding. Here we reported a comprehensive transcriptomics study to capture the global gene profiles during floral bud development in C. azalea. We examined the genome-wide gene expression between three developmental stages including floral bud initiation, floral organ differentiation and bud outgrowth, and identified nine co-expression clusters with distinctive patterns. Further, we identified the differential expressed genes (DEGs) during development and characterized the functional properties of DEGs by Gene Ontology analysis. We showed that transition from floral bud initiation to floral organ differentiation required changes of genes in flowering timing regulation, while transition to floral bud outgrowth was regulated by various pathways such as cold and light signaling, phytohormone pathways and plant metabolisms. Further analyses of dormancy associated MADS-box genes revealed that SVP- and AGL24- like genes displayed distinct expression patterns suggesting divergent roles during floral bud development.

Distinct double flower varieties in Camellia japonica exhibit both expansion and contraction of C-class gene expression.

BackgroundDouble flower domestication is of great value in ornamental plants and presents an excellent system to study the mechanism of morphological alterations by human selection. The classic ABC model provides a genetic framework underlying the control of floral organ identity and organogenesis from which key regulators have been identified and evaluated in many plant species. Recent molecular studies have underscored the importance of C-class homeotic genes, whose functional attenuation contributed to the floral diversity in various species. Cultivated Camellia japonica L. possesses several types of double flowers, however the molecular mechanism underlying their floral morphological diversification remains unclear.ResultsIn this study, we cloned the C-class orthologous gene CjAG in C. japonica. We analyzed the expression patterns of CjAG in wild C. japonica, and performed ectopic expression in Arabidopsis. These results revealed that CjAG shared conserved C-class function that controls stamen and carpel development. Further we analyzed the expression pattern of CjAG in two different C. japonica double-flower varieties, `Shibaxueshi’ and `Jinpanlizhi’, and showed that expression of CjAG was highly contracted in `Shibaxueshi’ but expanded in inner petals of `Jinpanlizhi’. Moreover, detailed expression analyses of B- and C-class genes have uncovered differential patterns of B-class genes in the inner organs of `Jinpanlizhi’.ConclusionsThese results demonstrated that the contraction and expansion of CjAG expression were associated with the formation of different types of double flowers. Our studies have manifested two different trajectories of double flower domestication regarding the C-class gene expression in C. japonica.

The APETALA1 and FRUITFUL homologs in Camellia japonica and their roles in double flower domestication

The APETALA1/FRUITFUL (AP1/FUL) family genes encode MADS-box transcription factors, which are broadly involved in many aspects of floral development in higher plants. Gene duplication in the core eudicots has produced the euAP1 and euFUL clades. It remains unclear how the functional divergence of this gene family occurred. Camellia japonica is a famous ornamental species which belongs to the Theaceae (Ericales) group. Artificial selection for aesthetic flowers in Camellia has resulted in a remarkable diversity of floral forms, and double flower is one of the most important traits which provides a valuable resource for studying the underlying domestication mechanism. Here we isolated two homologs of the AP1/FUL family, named as CjAPL1 and CjAPL2, from C. japonica. Sequence and phylogenic analyses revealed that they were orthologs of FUL and AP1, respectively. We showed by gene expression profiling and ectopic expression in Arabidopsis that CjAPL1 and CjAPL2 potentially played different roles during floral development. Overexpression of CjAPL1/2 displayed similar phenotypes in Arabidopsis including early flowering, formation of terminal flowers, and increase in stamen and pistil numbers, but only in plants overexpressing CjAPL2 was the petal number increased. This therefore indicates that duplication of AP1- and FUL-like genes was functionally divergent in Ericales. Furthermore, higher expression levels of CjAPL1/2 were identified in four different double-flower varieties compared to wild single-flower camellias. Our results provide evidence for functional diversification of AP1-like and FUL-like genes in core eudicot species and point to their roles in double flower domestication.

Overexpression of phosphoenolpyruvate carboxylase from Jatropha curcas increases fatty acid accumulation in Nicotiana tabacum

Jatropha curcas L. is an excellent biofuel crop, which displays a high efficiency of carbon absorption, and seed oil of Jatropha can be efficiently processed to produce high-quality biodiesel. Plant phosphoenolpyruvate carboxylases (PEPCs) play important roles not only in initial fixation of atmospheric CO2 in C4 and Crassulacean acid metabolism (CAM) plants, but also in fatty acid biosynthesis in seeds of oil plants by regulating carbon partitioning. Here, we identified JcPEPC1 from J. curcas L. by homology cloning, and alignment analysis of protein sequence revealed JcPEPC1 was a plant C3-type PEPC, and shared high similarity to PEPC of castor oil plant Ricinus communis. We implemented detailed functional characterization of JcPEPC1 by expression analysis and transgenic tobacco. JcPEPC1 gene expressed in the leaves and seeds of J. curcas L., and remarkable increase of expression level was also detected at seed oil-accumulating stages. We overexpressed JcPEPC1 in tobacco, and showed the enzymatic activity of PEPC in transgenic plants was notably higher than wild type. Gas chromatography (GC) analysis elucidated the composition and total content of fatty acids were also altered. This study indicated JcPEPC1 played a fundamental role in fatty acid biosynthesis in Jatropha seeds. Our results proposed enhanced PEPC activity of Jatropha could improve biosynthesis of fatty acid, which implied critical functions in primary metabolism of non-photosynthetic PEPC.

Global gene expression defines faded whorl specification of double flower domestication in Camellia

Double flowers in cultivated camellias are divergent in floral patterns which present a rich resource for demonstrating molecular modifications influenced by the human demands. Despite the key principle of ABCE model in whorl specification, the underlying mechanism of fine-tuning double flower formation remains largely unclear. Here a comprehensive comparative transcriptomics interrogation of gene expression among floral organs of wild type and “formal double” and “anemone double” is presented. Through a combination of transcriptome, small RNA and “degradome” sequencing, we studied the regulatory gene expression network underlying the double flower formation. We obtained the differentially expressed genes between whorls in wild and cultivated Camellia. We showed that the formation of double flowers tends to demolish gene expression canalization of key functions; the faded whorl specification mechanism was fundamental under the diverse patterns of double flowers. Furthermore, we identified conserved miRNA-targets regulations in the control of double flowers, and we found that miR172-AP2, miR156-SPLs were critical regulatory nodes contributing to the diversity of double flower forms. This work highlights the hierarchical patterning of global gene expression in floral development, and supports the roles of “faded ABC model” mechanism and miRNA-targets regulations underlying the double flower domestication.

Overexpression of CaAPX Induces Orchestrated Reactive Oxygen Scavenging and Enhances Cold and Heat Tolerances in Tobacco

Ascorbate peroxidase (APX) acts indispensably in synthesizing L-ascorbate (AsA) which is pivotal to plant stress tolerance by detoxifying reactive oxygen species (ROS). Enhanced activity of APX has been shown to be a key step for genetic engineering of improving plant tolerance. However it needs a deeper understanding on the maintenance of cellular ROS homeostasis in response to stress. In this study, we identified and characterized an APX (CaAPX) gene from Camellia azalea. Quantitative real-time PCR (qRT-PCR) analysis showed that CaAPX was expressed in all tissues and peaked in immature green fruits; the expression levels were significantly upregulated upon cold and hot stresses. Transgenic plants displayed marked enhancements of tolerance under both cold and heat treatments, and plant growth was correlated with CaAPX expression levels. Furthermore, we monitored the activities of several ROS-scavenging enzymes including Cu/Zn-SOD, CAT, DHAR, and MDHAR, and we showed that stress tolerance was synchronized with elevated activities of ROS-scavenging. Moreover, gene expression analysis of ROS-scavenging enzymes revealed a role of CaAPX to orchestrate ROS signaling in response to temperature stresses. Overall, this study presents a comprehensive characterization of cellular response related to CaAPX expression and provides insights to breed crops with high temperature tolerances.

Comparative genomics analysis reveals gene family expansion and changes of expression patterns associated with natural adaptations of flowering time and secondary metabolism in yellow Camellia

Yellow-flowering species are unique in the genus Camellia not only for their bright yellow pigments but also the health-improving substances in petals. However, little is known regarding the biosynthesis pathways of pigments and secondary metabolites. Here, we performed comparative genomics studies in two yellow-flowered species of the genus Camellia with distinctive flowering periods. We obtained 112,190 and 89,609 unigenes from Camellia nitidissima and Camellia chuongtsoensis, respectively, and identified 9547 gene family clusters shared with various plant species and 3414 single-copy gene families. Global gene expression analysis revealed six comparisons of differentially expressed gene sets in different developmental stages of floral bud. Through the identification of orthologous pairs, conserved and specific differentially expressed genes (DEGs) between species were compared. Functional enrichment analysis suggested that the gibberellin (GA) biosynthesis pathway might be related to the alteration of flowering responses. Furthermore, the expression patterns of secondary metabolism pathway genes were analyzed between yellow- and red-flowered Camellias. We showed that the key enzymes involved in glycosylation of flavonoids displayed differential expression patterns, indicating that the direct glycosylation of flavonols rather than anthocyanins was pivotal to coloration and health-improving metabolites in the yellow Camellia petals. Finally, the gene family analysis of UDP-glycosyltransferases revealed an expansion of group C members in C. nitidissima. Through comparative genomics analysis, we demonstrate that changes of gene expression and gene family members are critical to the variation of natural traits. This work provides valuable insights into the molecular regulation of trait adaptations of floral pigmentation and flowering timing.

The potential role of B-function gene involved in floral development for double flowers formation in Camellia changii Ye

Camellia changii Ye, a rare and endangered species, has a phenotype that sepals frequently transform into petals. We assumed that this change would cause single C. changii Ye turned double flowers and this was confirmed by the double flowers we found in grafted C. changii Ye. The microstructure of floral organs showed that: in perfect petals, the anthocyanin is distributed in the upper and lower epidermis; in petaloid sepals, anthocyaninin had both sepal and petal identities and sepals gradually transformed into petals; in spot-petaloid sepals, anthocyaninin is only distributed in upper epidermis. B-function gene GLOBOSA1 (GLO1) and GLOBOSA2 (GLO2) had high expression at the part with petal identity and had low or no expression at or near the part with sepal identity and these kinds of expression showed that gene played an important role in determining petal identity. B-function gene DEFICIENS (DEF) and TOMATO MADS BOX GENE6 (TM6) had high expression at the fused part of the stamens and this implied the importance of gene when stamen is transformed into petal. Thus, B-function gene is very important when C. changii Ye evolved into double flowers. Key words: Petaloid phenotype, double flowers, B-function gene, Camellia changii Ye, real-time polymerase chain reaction (PCR).

Unraveling the Roles of Regulatory Genes during Domestication of Cultivated Camellia: Evidence and Insights from Comparative and Evolutionary Genomics

With the increasing power of DNA sequencing, the genomics-based approach is becoming a promising resolution to dissect the molecular mechanism of domestication of complex traits in trees. Genus Camellia possesses rich resources with a substantial value for producing beverage, ornaments, edible oil and more. Currently, a vast number of genetic and genomic research studies in Camellia plants have emerged and provided an unprecedented opportunity to expedite the molecular breeding program. In this paper, we summarize the recent advances of gene expression and genomic resources in Camellia species and focus on identifying genes related to key economic traits such as flower and fruit development and stress tolerances. We investigate the genetic alterations and genomic impacts under different selection programs in closely related species. We discuss future directions of integrating large-scale population and quantitative genetics and multiple omics to identify key candidates to accelerate the breeding process. We propose that future work of exploiting the genomic data can provide insights related to the targets of domestication during breeding and the evolution of natural trait adaptations in genus Camellia.