Nitric oxide induced carotenoid contents in Crocus sativus under salinity

Abstract Crocus sativus possesses unique apocarotenoid compounds such as crocins and picrocrocein involved in color, taste, flavor and medicinal benefits of Saffron. Crocus sativus L. corms were treated with Nitric Oxide (NO) and salt. Crocins and picrocrocin contents were determined with high-performance liquid chromatography (HPLC) resulted a significant increase of crocins in treated plants with NO and salt that reveals the stimulating effect of NO in apocarotenoid biosynthesis besides the inductive role of salt stress. This raise can be attributed to expression of CsPDS, CsPSY, CsLYC, CsBCH, and CsCCD2 that were remarkably altered. Treating plants with NO caused more phenol production in oppose to less flavonoid content; however, salinity could increase both. Therefore, NO induced crocins and picrocrocin biosynthesis due to impressing gene expression. This increasing effect was enhanced when salinity was simultaneously imposed.


Introduction
Plant secondary metabolites are often considered crucial for plant responses against stress and adaptation. In different plant species a wide range of secondary metabolites are synthesized from primary metabolites. Secondary metabolites have fundamental roles in plant taste, color and odor, so they are regarded as sources of flavors, food additives and pharmaceutical compounds (Ramakrishna and Ravishankar 2011). Crocus sativus is a triploid, sterile plant that has been utilized as a medicinal plant and a spice for thousands of years. Iran, India, Greece, Morocco, Azerbaijan, Afghanistan and Spain are the main countries in Crocus sativus production. This plant's stigma is the source of a wide range of carotenoids and some sort of apocarotenoids that are exclusively produced in Crocus such as crocins and picrocrocin, color and flavor provider of the most expensive flavor "Saffron". Also, these apocarotenoids have pharmacological properties (Baba and Ashraf 2016). Moreover, saffron is a rich source of bio-molecules such as polyphenols and antioxidants (Tuberoso et al. 2016).
Crocins and picrocrocin are synthesized through Mevalonic-acid pathway. Carotenoids, are derived from the ubiquitous C5 building blocks isopentenyl diphosphate and dimethylallyl diphosphate. At the first step of this pathway three molecules of acetyl coenzyme A form isopentenyl pyrophosphate (IPP), then two molecules of geranyl geranyl pyrophosphate (GGPP) form phytoene by activity of Phytoene synthase (PSY), by phytoene desaturase (PDS) activity phytoene changes to lycopene. Lycopene beta-cyclase (LYC) activity caused b-carotene production. b-carotene hydroxylase enzyme (BCH) carries out the b-carotene hydroxylation for producing zeaxanthin (Castillo et al. 2005). Crocins are generated from the cleavage of zeaxanthin at the 7,8/ 7 0 ,8 0 positions by a carotenoid cleavage dioxygenase (CCD2) to produce picrocrocin and crocetin dialdehyde that is dehydrogenated and glycosylated to crocins (Frusciante et al. 2014). Recent studies on the changes of the secondary metabolites in saffron have demonstrated that the transcription regulation of the biosynthesis of crocins and safranal is controlled by genes CsBCH, CsZCD, and CsLYC (Ahrazem et al. 2010). The expression profile of CsLYC, CsBCH shows a correlation with the biosynthesis of crocins and safranal. In recent years, it has been reported that, when plants subjected to various environmental stresses, elicitors, or signal molecules, secondary metabolites accumulate (Taherkhani et al. 2017) and Nitric oxide a biologically active gas is able to regulate a variety of physiological and developmental processes (Wu et al. 2007). In addition to its signalling roles, it may act as a regulator for gene expression (Qiao and Fan 2008). The target of the present study is investigating the effect of exogenous NO and salinity on Crocus sativus stigmas apocarotenoids, biomolecule contents and expression of CsPSY, CsPDS, CsBCH, CsLYC and CsCCD2, involved in the carotenoid biosynthesis pathway.

Results and discussion
Colorimetric methods obtained results revealed that phenol and flavonoid contents got doubled in contrast with b-carotene and lycopene amounts that are reduced remarkably under treatments (Figure 1). Walia et al. (2005) reported an increase in the flavonoid biosynthesis pathway in rice during salinity too. Babu et al. (2011) reported that salt stress inhibits the expression of the gene encoded lycopene b-cyclase that converts lycopene to beta carotene. The decrease in lycopene and b-carotene may relate to photosynthesis reduction caused by salinity, "due to the fact that salinity can inhibit or upregulate the biosynthetic pathway of carotenoids through inhibition of CsLYC gene" (Dumas et al. 2003). The carotenoid pathway gene expressions are affected by abiotic stresses such as light, water availability, high temperature, salinity, cold and frosty conditions (Moshtaghi et al. 2010).
HPLC analysis results showed an increase of the crocins content under NO and salinity treatments;however, picrocrocin content raised under NO treatment and also NO þ salinity treatment (Table 1). Taherkhani et al. (2017) also reported an increasing trend in crocins and safranal contents of saffron plants under ultrasound treatment. It has been found that this raise is related to expression of the carotenoid biosynthesis genes.
The expression of involved genes in the carotenoid biosynthesis pathway was highly impressed by treating Crocus sativus plants with salt and NO (Table 2). CsPSY expressed in plants that were treated with NO þ100 mM NaCl by far more than other samples. Similarly, CsPDS was expressed in these plants more than other treatments. The most CsBCH expression was observed in plants that were treated with NO and received NaCl same as CsLYC. CsCCD2 that was expressed in NO þ 50mM NaCl more than other treatments even NO þ 100 mM NaCl. CsBCH and CsPDS play crucial roles in crocetin and crocins production in saffron. They are key enzymes in carotenoid production chain reaction (Fernandez 2004). We observed that in NO þ 50 mM NaCl