Platycladus orientalis is a tree species that is highly resistant, widely adaptable, and long-lived, with lifespans of even thousands of years. To explore the mechanisms underlying these characteristics, gene expressions have been investigated at the transcriptome level by RNA-seq combined with a digital gene expression (DGE) technique. So, it is crucial to have a reliable set of reference genes to normalize the expressions of genes in P. orientalis under various conditions using the most accurate and sensitive method of quantitative real-time PCR (qRT-PCR). In this study, we selected 10 reference gene candidates from transcriptome data of P. orientalis, and examined their expression profiles by qRT-PCR using 29 different samples of P. orientalis, which were collected from plants of different ages, different tissues, and plants subjected to different treatments including cold, heat, salinity, polyethylene glycol (PEG), and abscisic acid (ABA). Three analytical software packages (geNorm, Bestkeeper, and NormFinder) were used to assess the stability of gene expression. The results showed that ubiquitin-conjugating enzyme E2 (UBC) and alpha-tubulin (aTUB) were the optimum pair of reference genes at all developmental stages and under all stress conditions. ACT7 was the most stable gene across different tissues and cold-treated samples, while UBQ was the most stably expressed reference gene for NaCl- and ABA-treated samples. In parallel, aTUB and UBC were used singly or in combination as reference genes to examine the expression levels of NAC (a homolog of AtNAC2) in plants subjected to various treatments with qRT-PCR. The results further proved the reliability of the two selected reference genes. Our study will benefit future research on the expression of genes in response to stress/senescence in P. orientalis and other members of the Cupressaceae.

ABSTRACTg-aminobutyric acid (GABA) is a four-carbon non-protein amino acid presented in a wide range of organisms. In this study, a suppression subtractive hybridization (SSH) library was constructed using roots of a legume shrub, Caragana intermedia, with the combined treatment of 300 mM NaCl and 300 mM NaCl + 10 mM GABA. We obtained 224 GABA-regulated unique expressed sequence tags (ESTs) including signal transduction, transcriptional regulation, hormone biosynthesis, reactive oxygen species (ROS) and polyamine metabolism, etc. The key H2O2-generated genes, NADPH oxidase (CaGR60), peroxidase (CaGR61) and amine oxidase (CaGR62), were regulated at the mRNA level by 10 mM GABA, which clearly inhibited H2O2 accumulation brought about by NaCl stress in roots and leaves with the observation of 3,3Ј-diaminobenzidine (DAB) staining. Similarly, 10 mM GABA also regulated the expression of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes (CaGR30 and CaGR31) and ethylene production in NaCl-treated roots. Surprisingly, these H2O2-generated genes were enhanced at the mRNA level by a lower concentration of GABA, at 0.25 mM, but not other alternative nitrogen sources, and endogenous GABA accumulated largely just by the application of GABA at either concentration. Our results further proved that GABA, as a signal molecule, participates in regulating the expression of genes in plants under salt stress.

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Quercus bawanglingensis Huang, Li et Xing, an endemic evergreen oak of the genus Quercus (Fagaceae) in China, is currently listed in the Red List of Chinese Plants as a vulnerable (VU) plant.No chloroplast (cp) genome information is currently available for Q. bawanglingensis, which would be essential for the establishment of guidelines for its conservation and breeding. In the present study, the cp genome of Q. bawanglingensis was sequenced and assembled into double-stranded circular DNA with a length of 161,394 bp. Two inverted repeats (IRs) with a total of 51,730 bp were identified, and the rest of the sequence was separated into two single-copy regions, namely, a large single-copy (LSC) region (90,628 bp) and a small single-copy (SSC) region (19,036 bp). The genome of Q. bawanglingensis contains 134 genes (86 protein-coding genes, 40 tRNAs and eight rRNAs). More forward (29) than inverted long repeats (21) are distributed in the cp genome. A simple sequence repeat (SSR) analysis showed that the genome contains 82 SSR loci, involving 84.15% A/T mononucleotides. Sequence comparisons among the nine complete cp genomes, including the genomes of Q. bawanglingensis, Q. tarokoensis Hayata (NC036370), Q. aliena var. acutiserrata Maxim. ex , Lithocarpus balansae (Drake) A. Camus (KP299291) and Castanea mollissima Bl. (HQ336406), demonstrated that the diversity of SC regions was higher than that of IR regions, which might facilitate identification of the relationships within this extremely complex family. A phylogenetic analysis showed that Fagus engleriana and Trigonobalanus doichangensis form the basis of the produced evolutionary tree. Q. bawanglingensis and Q. tarokoensis, which belong to the group Ilex, share the closest relationship. The analysis of the cp genome of Q. bawanglingensis provides crucial genetic information for further studies of this vulnerable species and the taxonomy, phylogenetics and evolution of Quercus. single-copy regions (LSC and SSC) [4,5]. Due to its uniparental inheritance, highly conserved structure, general lack of recombination and small effective population size, the analysis of cp DNA has been deemed a useful method for evolution research and the exploration of plant systematics [6][7][8][9]. In fact, the availability of sufficient data on cp genomes is crucial for phylogenetic relationship reconstruction, i.e., the assessment of relationships within angiosperms [10][11][12], the identification of members of Pinaceae [13] and Pinus [14], and adequate comparisons, i.e., cp genomes from sister species [15] and possibly multiple individuals [16]. At present, approximately 3000 plastid genomes of Eukaryota are shareable in the National Center for Biotechnology Information database (NCBI; Available online: https: //www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?opt=plastid&taxid=2759&sort=Genome) due to improvements in sequencing technologies. In addition, molecular genetic methodologies based on nuclear and organellar genomes are crucial for conservation studies [17], particularly the con...