Transcriptome differences between 20- and 3, 000-year-old Platycladus orientalis reveal that ROS are involved in senescence regulation

Background: Platycladus orientalis has an extremely long life span of several thousands of years, attracting great interests in the mechanisms involved in such successful senescence regulation and resistance at physiological and molecular levels. Results: The levels of reactive oxygen species (ROS) were higher in 3,000-year-old than in 20-year-old P. orientalis, and the activities of GR and GSH demonstrated the same trend. We produced and analyzed massive sequence information from pooled samples of P. orientalis through transcriptome sequencing, which generated 51,664 unigenes with an average length of 475 bp. We then used RNA-seq analysis to obtain a high-resolution age­course profile of gene expression in 20- and 3,000-year-old P. orientalis individuals. Totally, 106 differentially expressed genes were obtained, of which 47 genes were downregulated and 59 upregulated in the old tree. These genes were involved in transcription factors, hormone-related responses, ROS scavengers, senescence-related responses, stress response, and defense and possibly play crucial roles in tackling various stresses in the 3,000-year-old P. orientalis during its life time. The expression patterns of genes related to ROS homeostasis further indicated that the high ability of ROS scavenging could be helpful for the 3,000-year-old P. orientalis to resist senescence. Conclusions: This study provides a foundation for the elucidation of senescence resistance through molecular studies and the discovery of useful genes in P. orientalis.

Isolation of high-quality RNA from Platycladus orientalis and other Cupressaceae plants

Platycladus orientalis has a lifespan of several thousand years in China, making it a good plant in which to study aging at the molecular level, but this requires sufficient quantities of high-quality P. orientalis RNA. However, no appropriate methods have been reported for total RNA isolation from P. orientalis leaves. The TRIzol method did not extract RNA, while cetyltrimethylammonium bromide, sodium dodecyl sulfate-phenol, and plant RNAout kit (Tianz, Inc., China) protocols resulted in low yields of poor quality RNA. Isolating total RNA using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, St. Louis, MO, USA) resulted in a high-quality product but a low yield. However, the two-step removal of polyphenols and polysaccharides in the improved plant RNAout kit protocol resulted in the isolation of RNA with a 28S:18S rRNA ratio of band intensities that was ~2:1, the A260/A280 absorbance ratio was 2.03, and the total RNA yield from P. orientalis leaves was high. This protocol was tested on different P. orientalis tissues of different ages and on leaves of five other Cupressaceae plants. The total RNAs were successfully used in complementary DNA synthesis for transcriptome sequencing and would be suitable to use in additional experiments. The results of this study will benefit future studies in Cupressaceae plants.

DNA sequence variation of drought-response candidate genes in Austrocedrus chilensis

Background: Austrocedrus chilensis (D. Don) Pic. Ser. et Bizzarri commonly known as Patagonian cypress is a member of the Cupressaceae family, characterized by a high adaptive potential for growing in marginal areas and good timber quality. The species grows over a wide area and under a wide range of rainfall. This study assessed adaptive genetic variation at SNP level in candidate genes involved in response to drought stress. Results: A total of 18 single nucleotide polymorphisms (SNPs) were found among 1,428 bp. Average nucleotide diversity value (π = 0.00312) was similar to those previously reported in other Cupressaceae. The Fst average among genes and populations was 0.163 and the lowest differentiation was observed in continuous and humid populations. A number of neutrality tests were applied to find evidence of positive selection in our candidate gene set, but only AcAQP2 gene in Pedregoso and San Ramón populations revealed significant departures from neutrality with positive values suggesting balancing selection. Conclusions: In this study we report the levels of nucleotide diversity searched in some drought stress candidate genes in Austrocedrus chilensis and the selective factors that may be acting on this species.