Protein domains with similarity to plant strictosidine synthase-like (SSL) sequences have been uncovered in the genomes of all multicellular organisms sequenced so far and are known to play a role in animal immune responses. Among several distinct groups of Arabidopsis thaliana SSL sequences, four genes (AtSSL4-AtSSL7) arranged in tandem on chromosome 3 show more similarity to SSL genes from Drosophila melanogaster and Caenorhabditis elegans than to other Arabidopsis SSL genes. To examine whether any of the four AtSSL genes are immune-inducible, we analysed the expression of each of the four AtSSL genes after exposure to microbial pathogens, wounding and plant defence elicitors using real-time quantitative RT-PCR, Northern blot hybridisation and Western blot analysis with antibodies raised against recombinant AtSSL proteins. While the AtSSL4 gene was constitutively expressed and not significantly induced by any treatment, the other three AtSSL genes were induced to various degrees by plant defence signalling compounds, such as salicylic acid, methyl jasmonate and ethylene, as well as by wounding and exposure to the plant pathogens Alternaria brassicicola and cucumber mosaic virus. Our data demonstrate that the four SSL-coding genes are regulated individually, suggesting specific roles in basal (SSL4) and inducible (SSL5-7) plant defence mechanisms.

The changes in the antioxidant enzymes activity, total protein and proline content and their correlations with freezing tolerance (FT) (expressed as LT 50 ) were investigated at 11 different olive cultivars at cold-acclimation (CA, in February) and non-acclimation (NA, in August) stages. Leaf samples were collected from each cultivar and were divided into two groups. The first group was immediately frozen in liquid nitrogen for further biochemical analysis. The second ones was subjected to different freezing temperatures (-5, -10, -15 and -20°C) for 10 h, in order to determine their FT. The unfrozen control samples were kept at 4°C. The results showed that Fishomi, Mission and Shengeh were the most freezing tolerant among other cultivars. In contrast, Zard, Manzanilla and Amigdalolia were the most sensitive ones. The cold acclimation enhanced the activities of superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), catalase (CAT), polyphenol oxidase (PPO) and total protein content. However, proline content and phenylalanine ammonia-lyase (PAL) activity did not change or even decreased slightly at CA stage, compare to those samples at NA stage. It was found that LT 50 to be closely correlated to POD, CAT, and PPO activity at CA and NA stages. Overall, higher leaf POD, CAT, and PPO activity could be used as important selection criteria in screening tolerant olive cultivars for cold zone climatic.

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Licorice is a well-known medicinal plant, containing various secondary metabolites of triterpenoid and phenolic families. The aim of this study is to evaluate the effect of salinity stress on the expression of key genes involved in the biosynthetic pathway of triterpenoids such as glycyrrhizin, betulinic acid, soyasaponins, and phytosterols in licorice root, as well as providing a phonemic platform to characterize antioxidant properties, glycyrrhizin, and total phenolic content. This study also includes measuring the gene expression level and glycyrrhizin content in leaves and roots of control plants. The studied genes included squalene synthase (SQS1 and SQS2), β-amyrin synthase (bAS), lupeol synthase (LUS), cycloartenol synthase (CAS), β-amyrin 11-oxidase (CYP88D6), and β-amyrin 24-hydroxylase (CYP93E6). Our results revealed that all of the mentioned genes were upregulated following the stress condition with different transcription rates. The highest increase (12-fold) was observed for the expression of the LUS gene, which is related to the betulinic acid pathway. Also, the highest content of glycyrrhizin was observed at 72 h posttreatment, which was consistent with the upregulated transcription levels of the glycyrrhizin pathway genes especially SQS1 and CYP88D6 at the same time. Correlation and stepwise regression analysis proved the key role of SQS1 gene in the biosynthetic pathway of glycyrrhizin. Antioxidant activity and phenolic content also were increased following stress condition. A comparison between the expression levels of SQS1 and other genes involved in the production of glycyrrhizin, phytosterols, and soyasaponins revealed a similar transcription trend, which shows the gene expression in the roots was significantly higher than the leaves. In contrast, SQS2 and LUS genes displayed a higher expression in leaf tissues. The genes related to betulinic acid biosynthetic pathway exhibited an expression rate different from other triterpenoid pathway genes, which could be observed in the leaves and roots of control plants and the roots of salt-treated plants. Furthermore, results showed that these two SQS genes have different expression rates due to different plant tissues (roots and leaves) and stress conditions. Importantly, in contrast to previous reports, we detected the glycyrrhizin in leaf tissues. This result may indicate the presence of a different genetic background in native Iranian licorice germplasm. Keywords Gene expression . Glycyrrhiza glabra . Glycyrrhizin . Quantitative real-time PCR . Secondary metabolites Abbreviations bAS β-Amyrin synthase CAS Cycloartenol synthase CYP88D6 β-Amyrin 11-oxidase CYP93E6 β-Amyrin 24-hydroxylase FDP Farnesyl diphosphate GA3 Gibberellic acid HPLC High-performance liquid chromatography LUS Lupeol synthase MeJA Methyl jasmonate OSCs Oxidosqualene cyclases QRT-PCR Quantitative reverse transcription PCR SQS Squalene synthase

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Monoterpenoid indole alkaloids (MIA) are a diverse class of secondary metabolites important for plant protection and are drugs for treating human diseases. Arabidopsis thaliana (L.) is not known to produce MIAs, yet its genome has 15 genes with similarity to the periwinkle (Catharanthus roseus (L.) G. Don) strictosidine synthase (STR) gene. Phylogenetic analysis of strictosidine synthase-like (SSL) proteins reveals four well supported classes of SSLs in Arabidopsis. To determine if Arabidopsis produces active strictosidine synthase, Arabidopsis protein extracts were assayed for enzymatic activity and cDNAs were expressed in Escherichia coli. Arabidopsis protein extracts from leaves and hairy roots do not make strictosidine at levels comparable to C. roseus, but they metabolise one substrate, secologanin, a precursor of strictosidine in other plant species, and produce an ‘unknown’ compound proposed to be a dimer of secologanic acid. Recombinant Arabidopsis proteins expressed in E. coli were not active STRs. Quantitative PCR analysis was performed on class A Ssls and showed they are upregulated by salt, ultraviolet light and salicylic acid treatment. RNAi mutants of Arabidopsis with reduced expression of all four class A Ssls, suggest that class A SSL proteins can modify secologanin. Gene expression and metabolomics data suggests that class A Ssl genes may have a role in plant protection.

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