Cathepsin B-like cysteine protease ApCathB negatively regulates cryo-injury tolerance in transgenic Arabidopsis and Agapanthus praecox.

Cell death is an inevitably cryo-injury in cell and tissue cryopreservation. The research on programmed cell death (PCD) in plant cryopreservation is still in its infancy. In this study, the survival rate of Agapanthus praecox embryogenic callus was significantly improved when the vitrification solution was added with 20 µM E-64, which is an inhibitor of cathepsin B. For further investigating the relation between cathepsin B and cryo-injury, the coding gene of cathepsin B, ApCathB was isolated and characterized. A subcellular localization assay showed that ApCathB was located in cytomembrane. Heterologous overexpression of ApCathB reduced the recovery rate during Arabidopsis seedlings cryopreservation from 29.56 % to 16.46 %. Transgenic seedlings lost most of cell viability in hypocotyl after dehydration and lead to aggravated cryo-injury. The reduced survival rate of ApCathB-overexpressing embryogenic callus of A. praecox further confirmed its negatively function in cryo-injury tolerance. In addition, the survival of ApCathB-overexpressing lines was almost rescued by E-64. TUNEL detection showed intensified signal and ROS was burst, especially for H2O2. Furthermore, VPE, Metacaspase 1, Cyp15a and AIF genes related to cell death regulation were remarkably up-regulated in ApCathB-overexpressing embryogenic callus during cryopreservation. Additionally, the expression level of genes regulating cell degradation was also elevated, indicating accelerated cell death caused by ApCathB-overexpressing. Taken together, this work verified that ApCathB negatively regulated the cryo-injury tolerance and cell viability through mediating the PCD event in plant cryopreservation. Significantly, cathepsin B has potential to be a target to improve survival rate after cryopreservation.

Relative expression of putative genes involved in galanthamine and other Amaryllidaceae alkaloids biosynthesis in Narcissus field and in vitro tissues.

The Narcissus pseudonarcissus cv. Carlton contains Amaryllidaceae alkaloids namely galanthamine, lycorine, homolycorine, narciclasine, which are noted for their pharmaceutical properties such as for the treatment of early to mid-stage Alzheimer's diseases, cancer, tumor etc. Alkaloid biosynthesis using plant in vitro systems has been considered as a tool for drug discovery and the pathways are starting to be understood but still far from complete. Therefore, the study was emphasized to observe the relative expressions of putative genes involved in the biosynthetic pathway leading to the Amaryllidaceae alkaloids in field grown bulbs and developing cell culture systems in Narcissus. MS media fortified with growth regulators were used for the development of tissue culture from Carlton twin-scale explants. MS medium with high auxin, 20 mg/l NAA was the best medium for callus growth and maintenance while media with low auxin, 4 mg/l NAA and MS basal media gave the maximum bulblets. Field tissues showed a higher amount of galanthamine content; i.e. basal plate (1050-1310 µg Gal/g FW) and bulb (980-1150 µg Gal/g FW) than the culture derived samples; callus (1.0-7.0 µg Gal/g FW) and bulblets (12-215 µg Gal/g FW) on a fresh weight (FW) basis. GC-MS chromatograms of samples under study also showed the presence of other important alkaloids i.e. lycorine, homolycorine, lycorenine, haemanthamine, crinamine, lycoramine and tazettine. RNA extracted from in vitro callus, bulblets and field grown bulb, basal plate were used for PCR to detect the relative expression of putative genes; P450, PAL, TYDC and NpO4OMT normalized to actin. The selected transcripts for P450s and TYDC were expressed in both field and in vitro tissues. Higher expressions of PAL were observed in calli than field samples. The expression of NpN4OMT was notably higher in field samples than in vitro tissues. Therefore, in vitro tissues could be a good source for the reproducible and easy extraction of alkaloids from plants.

Cloning and characterization of ApCystatin, a plant cystatin gene from Agapanthus praecox ssp. orientalis responds to abiotic stress.

Plant cystatins are involved in the regulation of protein turnover and play important roles in defense mechanisms. We cloned the ApCystatin gene from Agapanthus praecox ssp. orientalis, a famous ornamental and medical plant. The complete cDNA sequence of ApCystatin is comprised of 1439 nucleotides with a 423 bp ORF encoding 140 amino acids. The mRNA level of ApCystatin was significantly up-regulated under various abiotic stress, such as salt, osmosis, oxidative and cold stresses, which suggested that ApCystatin participated in the plant's resistance to stress. The recombinant ApCystatin fusion protein expressed in E. coli transetta (DE3) cells was approximate 18 kDa. 25 µg of ApCystatin inhibited more than 95% activity of papain, suggesting ApCystatin as a papain-like protease inhibitor. As an exogenous substance, 1.60 µg/mL ApCystatin protein improved the regrowth percentage of Arabidopsis 60-h seedlings after cryopreservation from 30% to 47%. In addition, the relative survival rate of A. praecox embryogenic callus after cryopreservation also increased for 30% with addition of 1.20 µg/mL ApCystatin protein. This indicated that ApCystatin performed protective property against cryoinjury to Arabidopsis 60-h seedlings and A. praecox embryogenic callus during cryopreservation. Under various abiotic stress conditions, the recombinant ApCystatin protein showed significant advantage in growth rates at NaCl, mannitol, PEG6000, cold, acidic and alkaline conditions, compared to control. In conclusion, ApCystatin as a new member of plant cystatins exhibited protective property against cryoinjury in plant cryopreservation and abiotic stress in E. coli.

O-acetylserine(thio)lyase (OAS-TL) molecular expression in Pancratium maritimum L. (Amaryllidaceae) under salt stress.

MAIN CONCLUSION: Different levels of salt stress affected the OAS-TL expression levels in Pancratium maritimum organs (bulb, leaf and root). A detailed method has been described for the identification of the conserved domain of the OAS-TL cDNA in sea daffodil given the scarce data available for the Amaryllidaceae family. Pancratium maritimum or sea daffodil (Amaryllidaceae) is a bulbous geophyte growing on coastal sands. In this study, we investigated the involvement of cysteine synthesis for salt tolerance through the expression of the enzyme O-acetylserine(thio)lyase (OAS-TL) during the stress response to NaCl treatments in P. maritimum. Quantitative real-time PCR was used in different organs (bulb, leaf and root).