Background: Even though real-time PCR has been broadly applied in biomedical sciences, data processing procedures for the analysis of quantitative real-time PCR are still lacking; specifically in the realm of appropriate statistical treatment. Confidence interval and statistical significance considerations are not explicit in many of the current data analysis approaches. Based on the standard curve method and other useful data analysis methods, we present and compare four statistical approaches and models for the analysis of real-time PCR data. Results: In the first approach, a multiple regression analysis model was developed to derive ΔΔCt from estimation of interaction of gene and treatment effects. In the second approach, an ANCOVA (analysis of covariance) model was proposed, and the ΔΔCt can be derived from analysis of effects of variables. The other two models involve calculation ΔCt followed by a two group t- test and non-parametric analogous Wilcoxon test. SAS programs were developed for all four models and data output for analysis of a sample set are presented. In addition, a data quality control model was developed and implemented using SAS. Conclusion: Practical statistical solutions with SAS programs were developed for real-time PCR data and a sample dataset was analyzed with the SAS programs. The analysis using the various models and programs yielded similar results. Data quality control and analysis procedures presented here provide statistical elements for the estimation of the relative expression of genes using real-time PCR.

Significance: Land plants produce numerous terpenoids that regulate development and mediate environmental interactions. Thus, how typical plant terpene synthase ( TPS ) genes originated and evolved to create terpenoid diversity is of fundamental interest. By investigating TPSs from the genomes and transcriptomes of diverse taxa of green plants, it was demonstrated here that the ancestral TPS gene originated in land plants after divergence from green algae and encoded a bifunctional ent -kaurene synthase for phytohormone biosynthesis. This ancestral TPS then underwent gene duplication at least twice early in land plant evolution, leading to three ancient TPS lineages reflecting sub-functionalization of class I and II activities for phytohormone biosynthesis and neo-functionalization from primary to secondary metabolism, followed in each case by dynamic functional divergence.

Summary: Rice plants fed on by fall armyworm (Spodoptera frugiperda, FAW) caterpillars emit a blend of volatiles dominated by terpenoids. These volatiles were highly attractive to females of the parasitoid Cotesia marginiventris. Microarray analysis identified 196 rice genes whose expression was significantly upregulated by FAW feeding, 18 of which encode metabolic enzymes potentially involved in volatile biosynthesis. Significant induction of expression of seven of the 11 terpene synthase (TPS) genes identified through the microarray experiments was confirmd using real‐time RT‐PCR. Enzymes encoded by three TPS genes, Os02g02930, Os08g07100 and Os08g04500, were biochemically characterized. Os02g02930 was found to encode a monoterpene synthase producing the single product S‐linalool, which is the most abundant volatile emitted from FAW‐damaged rice plants. Both Os08g07100 and Os08g04500 were found to encode sesquiterpene synthases, each producing multiple products. These three enzymes are responsible for production of the majority of the terpenes released from FAW‐damaged rice plants. In addition to TPS genes, several key genes in the upstream terpenoid pathways were also found to be upregulated by FAW feeding. This paper provides a comprehensive analysis of FAW‐induced volatiles and the corresponding volatile biosynthetic genes potentially involved in indirect defense in rice. Evolution of the genetic basis governing volatile terpenoid biosynthesis for indirect defense is discussed.

As a response to caterpillar feeding, poplar releases a complex mixture of volatiles which comprises several classes of compounds. Poplar volatiles have been reported to function as signals in plant-insect interactions and intra- and inter-plant communication. Although the volatile blend is dominated by mono- and sesquiterpenes, there is much to be learned about their formation in poplar. Here we report the terpene synthase (TPS) gene family of western balsam poplar (Populus trichocarpa) consisting of 38 members. Eleven TPS genes (PtTPS5-15) could be isolated from gypsy moth (Lymantria dispar)-damaged P. trichocarpa leaves and heterologous expression in Escherichia coli revealed TPS activity for ten of the encoded enzymes. Analysis of TPS transcript abundance in herbivore-damaged leaves and undamaged control leaves showed that seven of the genes, PtTPS6, PtTPS7, PtTPS9, PtTPS10, PtTPS12, PtTPS13 and PtTPS15, were significantly upregulated after herbivory. Gypsy moth-feeding on individual leaves of P. trichocarpa trees resulted in induced volatile emission from damaged leaves, but not from undamaged adjacent leaves. Moreover, the concentration of jasmonic acid and its isoleucine conjugates as well as PtTPS6 gene expression were exclusively increased in the damaged leaves, suggesting that no systemic induction occurred within the tree. Our data indicate that the formation of herbivore-induced volatile terpenes in P. trichocarpa is mainly regulated by transcript accumulation of multiple TPS genes and is likely mediated by jasmonates. The specific local emission of volatiles from herbivore-damaged leaves might help herbivore enemies to find their hosts or prey in the tree canopy.