Effects of Penicillium infection on the expression and activity of CDPK2 in postharvest Hami melon treated with calcium chloride
Introduction
Hami melon (Cucumis melo var. Saccharinus) is an economically important fruit crop in Xinjiang, China. However, pathogen infection and mechanical disruption can lead to postharvest Hami melon quality deterioration, negatively affecting sales and storage. In particular, Penicillium pathogens cause decay and deterioration of postharvest Hami melon in cold storage [1]. It has been shown that treatment with hot water and shellac can reduce the incidence of Fusarium spp. and Alternaria spp. infection in Hami melon and increase the activity of defensive enzymes in some fruits [2]. Natamycin combined with the bilayer membrane composed of chitosan and polyethylene wax can inhibit the growth of Alternaria spp. and semisolid Fusarium spp. on the surface of muskmelon [3]. Although many studies have been done on the disease resistance of postharvest Hami melon, there is little research on Penicillium infection. It is of great significance for postharvest storage of Hami melon to study the defence mechanism(s) of Hami melon against Penicillium infection.
Calcium ions (Ca2+) are second messengers in plant signal transduction involved in growth and development and the response to stress. Calcium-dependent protein kinase (CDPK) is a receptor for Ca2+ that plays an active role in plant disease resistance [[4], [5], [6]]. The inward movement of Ca2+ is a key early step in resistance-associated signalling pathways in plant cells infected by pathogenic microorganisms. As an important downstream receptor of Ca2+ in plants, CDPK may play a role in plant disease resistance [[7], [8], [9], [10]]. It has been shown that treatment with calcium chloride (concentration of calcium ions >0.16 and ≤ 0.08 mol/L) can accelerate the aging of muskmelo [11]. Comparison of different concentrations of calcium ions (0.08, 0.16, and 0.24 mol/L) showed that treatment with exogenous 0.16 mol/L calcium delayed fruit senescence and improved fruit quality [12].
CDPK is a serine/threonine-type protein kinase (CPK) present in plants and some protozoa, but not in animals [[13], [14], [15], [16]]. CDPK is a multifunctional protein with an N-terminal variable domain, a kinase domain, an autoinhibitory domain, and a calmodulin-like domain [[17], [18], [19]]. The CDPK gene is expressed in plants such as rice, Arabidopsis, grape, red pepper, and peanut [[20], [21], [22], [23], [24]], and is involved in plant responses to stress. In Arabidopsis, plants overexpressing AtCPK1 display broad-spectrum resistance to bacteria and fungi [25], and overexpression of NtCDPK2 inhibits the synthesis of salicylic acid and the expression of PR1a and PR2a that relate to diseases regulated by salicylic acid [26]. In grape, some CDPK genes (VpCDPK6, VpCDPK9, VpCDPK14, VpCDPK16, and VpCDPK19) are upregulated in response to the powdery mildew pathogen [27], which also induces the expression of the TaCPK2 gene in wheat [28], but the CaCDPK10 gene was downregulated in response to Ralstonia solanacearum in pepper [29].
CDPKs have been extensively studied in many plant species at both the gene and protein level, but not in Hami melon. During the storage and transportation of this melon, Penicillium infection causes severe economic losses. In the present study, expression of the HmCDPK2 gene in Hami melon was investigated before and after Penicillium infection by measuring CDPK activity, sequence analysis, phylogenetic tree construction, prediction of the secondary and tertiary structure of the HmCDPK2 protein, and fluorescence quantitative PCR. The results provide useful physiological and molecular biological information relevant to the storage and preservation of Hami melon.
Section snippets
Penicillium spore suspension
The Penicillium (Penicillium crustosum FRR 1669T) strain used in this study was isolated from Hami melon surface in previous study by our lab. The Penicillium strain was grown in the potato medium (2% glucose, 2% agar, 20% potato) and then cultivated at 28 °C until a large quantity of spores were produced. The Penicillium spores were harvested by using sterile water flushing and the spores suspensions were diluted with sterile water to obtain 1 × 105 spores/mL dilutions.
Plant material and fruit treatment
The Jiashi Hami melons
HmCDPK2 sequence analysis and phylogenetic tree construction
Database analysis using the determined sequence identified Unigene4321_QHMG, with a length of 1635 bp, and ORF Finder found the largest ORF of 1239 nucleotides, with ATG and TAG initiation as stop codons, respectively, encoding a 412 amino acid polypeptide (Fig. 1). HmCDPK2 from Hami melon shares 99% sequence identity with XP_004135049.2 in cucumber, 96% with AAB49984.1 in Cucurbita pepo, 89% with XP_010090475.1 in Morus notabilis and AGS14999.1 in Vitis amurensis, 88% with EOY19314.1 in cocoa,
Discussion
The CDPK gene family has a long evolutionary history dating back to ancient terrestrial plants such as ferns and mosses that contain multiple CDPK genes in their genomes [30]. In the present study, the amino acid sequence of HmCDPK2 in Hami melon was aligned with those in other species. The results showed that Hami melon and cucumber belong to the same branch, and the Hami melon sequence shares higher sequence homology with Cucurbita pepo and lower sequence homology with Morus notabilis and
Conclusion
Hami melon can, after treatment with a suitable concentration of calcium ions (0.16 M), resist Penicillium infection by inducing CDPK activity and upregulating expression of the HmCDPK2 gene. The response of CDPK to Penicillium mainly manifested in the early phase of infection. This experiment has a preliminary understanding of the relationship between HmCDPK2 gene expression and enzyme activity under different concentrations of CaCl2 solution, and provides a new theoretical basis for the
Acknowledgements
This work was supported by the National Natural Science Foundation of China (31360412) and the High-level Talents Scientific Research Project of Shihezi University (RCZ201540).
References (37)
Reduction in Hami melon ( Cucumis melo, var. saccharinus ) softening caused by transport vibration by using hot water and shellac coating
Postharvest Biol. Technol.
(2015)- et al.
Use of surface coatings with natamycin to improve the storability of Hami melon at ambient temperature
Postharvest Biol. Technol.
(2007) - et al.
Calcium oscillations in higher plants
Curr. Opin. Plant Biol.
(2001) - et al.
CDPKs in immune and stress signaling
Trends Plant Sci.
(2013) Signal transmission in the plant immune response
Trends Plant Sci.
(2001)Calcium alters senescence rate of postharvest muskmelon fruit disks
Postharvest Biol. Technol.
(1996)- et al.
Biochemical regulation of in vivo function of plant calcium-dependent protein kinases (CDPK)
Biochim. Biophys. Acta
(2013) - et al.
Expression of calcium-dependent protein kinase (CDPK) genes under abiotic stress conditions in wild-growing grapevine Vitis amurensis
J. Plant Physiol.
(2013) - et al.
Ancient signals: comparative genomics of green plant CDPKs
Trends Plant Sci.
(2014) - et al.
Expression of rice Ca 2+ -dependent protein kinases (CDPKs) genes under different environmental stresses
FEBS Lett.
(2007)