Cysteine Protease Gene Research Articles

Identification and phylogenetic of cysteine peptidase genes from the bamboo pest Hippotiscus dorsalis, based on RNA-seq and PacBio Iso-Seq

One of the insects that cause the most damage to Moso bamboos in the southern regions of China is Hippotiscus dorsalis (Hemiptera: Pentatomidae). However, the study of molecular and growth development analysis is limited because its genetic resources are still in the blank stage. In order to improve our knowledge of the molecular traits of this insect pest, we first generated the transcriptome from H dorsalis using PacBio Iso-Seq. With an average length of 1495 bp, 207,663 nonredundant full-length reads were produced. Using five databases, we annotated 36,238 transcripts: Uniprot (24,388), GO (1160), NR (24,110), Pfam (9333), and KEGG (14,497). According to aligning of each transcript in the NR database, most of them (85.147 %) were annotated into the functional genes from Halyomorpha halys. In the meantime, we discovered 33,689 long noncoding RNAs (lncRNAs), 4408 transcription factors (TFs), and 586 alternative splicing (AS) events, which involved in transcriptional regulation. Additionally, the candidate cathepsin genes from the bamboo pest were examined in order to comprehend how they adapted to their host. To sum up, our findings offer a fresh resource for comprehensive transcriptional data and insights into the genetics and gene expression of H dorsalis.

Papain-like cysteine protease gene family in fig (Ficus carica L.): genome-wide analysis and expression patterns

Papain-like cysteine protease gene family in fig (<i>Ficus carica</i> L.): genome-wide analysis and expression patterns

Genome-Wide Identification and Male Sterility-Related Expression Analysis of Papain-like Cysteine Protease Gene Family in Capsicum annuum

PLCPs (papain-like cysteine proteases) are one of the most abundant groups of cysteine proteases and play vital roles in multiple processes. The pepper (Capsicum annuum) is an important Solanaceae vegetable crop; its commercial hybrid seeds are widely used in production. Male sterility is a valuable trait for hybrid seed production. However, the function of PLCPs and the underlying mechanisms of male sterility in peppers remain unclear. In this study, we comprehensively identified the PLCP gene family in peppers, identifying 31 CaPLCPs. A phylogenetic analysis classified 31 members into eight clades. These CaPLCPs were unevenly distributed across eight chromosomes, and five segmental duplicated pairs were observed. The promoter cis-acting element analysis indicated that CaPLCP promoters contained abundant hormone-responsive and stress-responsive cis-elements, suggesting that CaPLCPs may play important roles in responding to abiotic stress, such as drought and low temperatures, as well as in plant immunity. The qRT-PCR analysis demonstrated that the expression levels of CaPLCP1, CaPLCP5, CaPLCP11, CaPLCP12, CaPLCP13, CaPLCP17, CaPLCP19, and CaPLCP21 were significantly reduced in the flowers of MS (male sterile pepper) at least at one stage, indicating their potential roles as regulatory factors in pepper male sterility. These findings provide important insights into the functional analysis of the PLCP gene family in peppers and other species, laying a crucial foundation for understanding the mechanisms of male sterility in peppers.

Indications of programmed cell death in wheat roots upon exposure to silver nanoparticles

Programmed cell death (PCD) can occur at every developmental stage as a plant’s response to various biotic and abiotic environmental factors. Silver nanoparticles (AgNPs) are widely used in consumer products and possess antimicrobial properties, making them important in assessing nanoparticle effects on plants. In the present study, we examined the impact of AgNPs (0, 0.5, 1, 5, 10, and 20 mg L-1) on wheat root PCD by evaluating parameters such as the mitotic index, chromosomal behaviors, nuclear deformation, cytochrome c release, caspase-1-like activity, and the expression of cysteine protease genes (TaVPE4, TaMCA1, and TaMCA4). Our findings revealed a dose-dependent decrease in the mitotic index ratio and increased chromosomal abnormalities induced by AgNPs. Additionally, we observed various hallmarks of PCD, including chromatin condensation, slight DNA smear, reduction in mitochondrial inner membrane potential, and cytochrome c release to the cytoplasm as well as increased caspase-1-like activity and TaVPE4 gene expression. Notably, the gene expressions of TaMCA1 and TaMCA4 were found to be antagonistically regulated by AgNPs, further indicating the induction of PCD by AgNP treatment. Overall, our study provides evidence of AgNP-induced PCD in wheat roots, elucidating the involvement of cysteine protease genes in this process.

Comparative analysis of cysteine proteases reveals gene family evolution of the group 1 allergens in astigmatic mites.

Astigmatic mites contain potent allergens that can trigger IgE-mediated immune responses, leading to allergic diseases such as asthma, allergic rhinitis and atopic dermatitis. In house dust mites Dermatophagoides pteronyssinus and Dermatophagoides farinae, group 1 allergens (Der p 1 and Der f 1), characterized as papain-like cysteine proteases, have been defined as the major allergens that have high prevalence and potency. Previous studies of mite group 1 allergens mainly focused on identification, comparison of sequence and structure, as well as the investigation of cross-reactivity. To achieve a comprehensive view of mite group 1 allergens, we performed a comparative genomic analysis of all the cysteine proteases in six astigmatic mite species to elucidate the evolutionary relationships of group 1 allergens. Based on the high-quality and annotated genomes, all the cysteine proteases in six astigmatic mite species were identified by sequence homology search. The phylogenetic relationships, gene synteny and expression levels were revealed by bioinformatic tools. The allergenicity of recombinant cysteine proteases was evaluated by enzyme-linked immunosorbent assay. Tandem duplication was revealed as the major feature of cysteine protease gene evolution in astigmatic mites. The high IgE-binding capacity and the significant expression level of the cysteine protease DP_007902.01 suggested its potential as a novel group 1 allergen of D. pteronyssinus. In addition, gene decay events were identified in the skin-burrowing parasitic mite Sarcoptes scabiei. This comprehensive analysis provided insights into the evolution of cysteine proteases, as well as the component-resolved diagnosis of mite allergies.

Molecular characterization and pathogenicity evaluation of enterovirus G isolated from diarrheic piglets.

Four enterovirus G (EV-G) strains were isolated from diarrheic piglets that were negative for common swine enteric viruses. The spherical enterovirus particles of roughly 30-nm diameter were observed under transmission electron microscopy by using plaque-purified enterovirus. The complete genome sequence analysis revealed that each of four enteroviruses contained a papain-like cysteine protease (PLCP) gene between the 2C and 3A junction regions of the viral genome. This insertion encoded a predicted protease similar to the PLCP of porcine torovirus. The phylogenetic analysis based on complete genome with and without PLCP gene revealed that the four isolated EV-G strains were grouped together with global enterovirus G1-PLCP strains, and more closely related to EV-G/PLCP strains previously detected in China, Japan, and Korea (90.3%-92.2% similarities based on nucleotides). The cell susceptibility test demonstrated that the isolated EV-G could infect and replicate in cell lines from various host species. Furthermore, pathogenicity evaluation showed that the isolated EV-Gs induced mild diarrhea, pyrexia, and reduced body weight in infected piglets. The epidemiological investigation revealed a high prevalence of EV-G in swine herds. Together, our findings demonstrate that the isolated EV-G is pathogenic in piglets and may be advantageous in providing more trustworthy data on the evolution and pathological properties of EV-G. IMPORTANCE Enterovirus G is a species of positive-sense single-stranded RNA viruses associated with several mammalian diseases. The porcine enterovirus strains isolated here were chimeric viruses with the PLCP gene of porcine torovirus, which grouped together with global EV-G1 strains. The isolated EV-G strain could infect various cell types from different species, suggesting its potential cross-species infection risk. Animal experiment showed the pathogenic ability of the isolated EV-G to piglets. Additionally, the EV-Gs were widely distributed in the swine herds. Our findings suggest that EV-G may have evolved a novel mechanism for broad tropism, which has important implications for disease control and prevention.

First Identification and Pathogenicity Evaluation of an EV-G17 Strain Carrying a Torovirus Papain-like Cysteine Protease (PLCP) Gene in China.

Enterovirus G (EV-G) is prevalent in pig populations worldwide, and a total of 20 genotypes (G1 to G20) have been confirmed. Recently, recombinant EV-Gs carrying the papain-like cysteine protease (PLCP) gene of porcine torovirus have been isolated or detected, while their pathogenicity is poorly understood. In this study, an EV-G17-PLCP strain, 'EV-G/YN23/2022', was isolated from the feces of pigs with diarrhea, and the virus replicated robustly in numerous cell lines. The isolate showed the highest complete genome nucleotide (87.5%) and polyprotein amino acid (96.6%) identity in relation to the G17 strain 'IShi-Ya4' (LC549655), and a possible recombination event was detected at the 708 and 3383 positions in the EV-G/YN23/2022 genome. EV-G/YN23/2022 was nonlethal to piglets, but mild diarrhea, transient fever, typical skin lesions, and weight gain deceleration were observed. The virus replicated efficiently in multiple organs, and the pathological lesions were mainly located in the small intestine. All the challenged piglets showed seroconversion for EV-G/YN23/2022 at 6 to 9 days post-inoculation (dpi), and the neutralization antibody peaked at 15 dpi. The mRNA expression levels of IL-6, IL-18, IFN-α, IFN-β, and ISG-15 in the peripheral blood mononuclear cells (PBMCs) were significantly up-regulated during viral infection. This is the first documentation of the isolation and pathogenicity evaluation of the EV-G17-PLCP strain in China. The results may advance our understanding of the evolution characteristics and pathogenesis of EV-G-PLCP.

High-Quality Genome Assembly and Genome-Wide Association Study of Male Sterility Provide Resources for Flax Improvement.

Flax is an economic crop with a long history. It is grown worldwide and is mainly used for edible oil, industry, and textiles. Here, we reported a high-quality genome assembly for "Neiya No. 9", a popular variety widely grown in China. Combining PacBio long reads, Hi-C sequencing, and a genetic map reported previously, a genome assembly of 473.55 Mb was constructed, which covers ~94.7% of the flax genome. These sequences were anchored onto 15 chromosomes. The N50 lengths of the contig and scaffold were 0.91 Mb and 31.72 Mb, respectively. A total of 32,786 protein-coding genes were annotated, and 95.9% of complete BUSCOs were found. Through morphological and cytological observation, the male sterility of flax was considered dominant nuclear sterility. Through GWAS analysis, the gene LUSG00017705 (cysteine synthase gene) was found to be closest to the most significant SNP, and the expression level of this gene was significantly lower in male sterile plants than in fertile plants. Among the significant SNPs identified in the GWAS analysis, only two were located in the coding region, and these two SNPs caused changes in the protein encoded by LUSG00017565 (cysteine protease gene). It was speculated that these two genes may be related to male sterility in flax. This is the first time the molecular mechanism of male sterility in flax has been reported. The high-quality genome assembly and the male sterility genes revealed, provided a solid foundation for flax breeding.

Modifying the Expression of Cysteine Protease Gene PCP Affects Pollen Development, Germination and Plant Drought Tolerance in Maize.

Cysteine proteases (CPs) are vital proteolytic enzymes that play critical roles in various plant processes. However, the particular functions of CPs in maize remain largely unknown. We recently identified a pollen-specific CP (named PCP), which highly accumulated on the surface of maize pollen. Here, we reported that PCP played an important role in pollen germination and drought response in maize. Overexpression of PCP inhibited pollen germination, while mutation of PCP promoted pollen germination to some extent. Furthermore, we observed that germinal apertures of pollen grains in the PCP-overexpression transgenic lines were excessively covered, whereas this phenomenon was not observed in the wild type (WT), suggesting that PCP regulated pollen germination by affecting the germinal aperture structure. In addition, overexpression of PCP enhanced drought tolerance in maize plants, along with the increased activities of the antioxidant enzymes and the decreased numbers of the root cortical cells. Conversely, mutation of PCP significantly impaired drought tolerance. These results may aid in clarifying the precise functions of CPs in maize and contribute to the development of drought-tolerant maize materials.

Modifying the expression of cysteine protease gene PCP affects pollen develop-ment, germination and plant drought tolerance in maize

Modifying the expression of cysteine protease gene PCP affects pollen develop-ment, germination and plant drought tolerance in maize

Modifying the expression of cysteine protease gene PCP affects pollen develop-ment, germination and plant drought tolerance in maize

Modifying the expression of cysteine protease gene PCP affects pollen develop-ment, germination and plant drought tolerance in maize

The Bcl-2-associated athanogene gene family in tobacco (Nicotiana tabacum) and the function of NtBAG5 in leaf senescence.

Leaf senescence in tobacco is closely related to leaf maturation and secondary metabolites. Bcl-2-associated athanogene (BAG) family members are highly conserved proteins and play key roles in senescence, growth and development, and resistance to biotic and abiotic stresses. Herein, the BAG family of tobacco was identified and characterized. In total, 19 tobacco BAG protein candidate genes were identified and divided into two classes, class I comprising NtBAG1a-e, NtBAG3a-b, and NtBAG4a-c and class II including NtBAG5a-e, NtBAG6a-b, and NtBAG7. Genes in the same subfamily or branch of the phylogenetic tree exhibited similarities in gene structure and the cis-element on promoters. RNA-seq and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) revealed that the expression of NtBAG5c-f and NtBAG6a-b was upregulated in senescent leaves, implying that they play a role in regulating leaf senescence. NtBAG5c was localized in the nucleus and cell wall as a homology of leaf senescence related gene AtBAG5. Further, the interaction of NtBAG5c with heat-shock protein 70 (HSP70) and sHSP20 was demonstrated using yeast two-hybrid experiment. Virus-induced gene silencing indicated that NtBAG5c reduced the lignin content and increased superoxide dismutase (SOD) activity and hydrogen peroxide (H2O2) accumulation. In NtBAG5c-silenced plants, the expression of multiple senescence-related genes cysteine proteinase (NtCP1), SENESCENCE 4 (SEN4) and SENESCENCE-ASSOCIATED GENE 12 (SAG12) was downregulated. In conclusion, tobacco BAG protein candidate genes were identified and characterized for the first time.

Novel recombinant porcine enterovirus G viruses lacking structural proteins are maintained in pig farms in Japan.

Type 1 recombinant enterovirus G (EV-G), which carries the papain-like cysteine protease (PLCP) gene of torovirus between its 2C/3A regions, and type 2 recombinant EV-G, which carries the torovirus PLCP gene with its flanking regions having non-EV-G sequences in place of the viral structural genes, have been detected in pig farms in several countries. In a previous study, we collected 222 fecal samples from 77 pig farms from 2104 to 2016 and detected one type 2 recombinant EV-G genome by metagenomics sequencing. In this study, we reanalyzed the metagenomic data and detected 11 type 2 recombinant EV-G genomes. In addition, we discovered new type 2 recombinant EV-G genomes of the two strains from two pig farms samples in 2018 and 2019. Thus, we identified the genomes of 13 novel type 2 recombinant EV-Gs isolated from several pig farms in Japan. Type 2 recombinant EV-G has previously been detected only in neonatal piglets. The present findings suggest that type 2 recombinant EV-G replicates in weaning piglets and sows. The detection of type 1 recombinant EV-Gs and type 2 recombinant EV-Gs at 3-year and 2-year intervals, respectively, from the same pig farm suggests that the viruses were persistently infecting or circulating in these farms.

Knockout of a papain-like cysteine protease gene OCP enhances blast resistance in rice.

Papain-like cysteine proteases (PLCPs) play an important role in the immune response of plants. In Arabidopsis, several homologous genes are known to be involved in defending against pathogens. However, the effects of PLCPs on diseases that afflict rice are largely unknown. In this study, we show that a PLCP, an oryzain alpha chain precursor (OCP), the ortholog of the Arabidopsis protease RD21 (responsive to dehydration 21), participates in regulating resistance to blast disease with a shorter lesion length characterizing the knockout lines (ocp-ko), generated via CRISPR/Cas9 technology. OCP was expressed in all rice tissues and mainly located in the cytoplasm. We prove that OCP, featuring cysteine protease activity, interacts with OsRACK1A (receptor for activated C kinase 1) and OsSNAP32 (synaptosome-associated protein of 32 kD) physically in vitro and in vivo, and they co-locate in the rice cytoplasm but cannot form a ternary complex. Many genes related to plant immunity were enriched in the ocp-ko1 line whose expression levels changed significantly. The expression of jasmonic acid (JA) and ethylene (ET) biosynthesis and regulatory genes were up-regulated, while that of auxin efflux transporters was down-regulated in ocp-ko1. Therefore, OCP negatively regulates blast resistance in rice by interacting with OsRACK1A or OsSNAP32 and influencing the expression profiles of many resistance-related genes. Moreover, OCP might be the cornerstone of blast resistance by suppressing the activation of JA and ET signaling pathways as well as promoting auxin signaling pathways. Our research provides a comprehensive resource of PLCPs for rice plants in defense against pathogens that is also of potential breeding value.

Molecular diversity for Entamoeba histolytica virulence factor genes isolated from patients with amoebic dysentery

Entamoebahistolytica is a parasitic protozoan that caused amoebiasis, a diseases which infects the liver and colon and spread all over the world. E.histolytica is causing the death of human cells and leading to develop invasion, and ulceration of the intestine. However, amoebic liver abscess(ALA) considers as major common extraintestinal disease caused by E.histolytica.The aims of this study, was to detect E. histolytica by polymerase chain reaction (PCR), and to differentiate it from other amoeba species in a stool samples of human. Study the pathogenicity of E. histolytica in human through determining the presence of virulence factors like cysteine protease, amoebapore and Gal/lectin. A total of 100 stool samples were collected from patients with amoebic dysentery and then examined microscopically to confirm the presence of E. histolytica and other Entamoeba species, 72 ( 72%) out of 100 examined samples with E.histolytica were positive by PCR and 28(28%) were negative. The positive samples with PCR have been submitted to detect the virulence factors of E.histolytica by PCR and the results showed that the total of 72(100%)were positive with cysteine proteases gene, shown 62(86.11%) were positive with amoebapore and 65(90.27) were positive with lectin. Gene sequence for cysteine protease, amoebapore and lectin in local E. histolytica human isolates showed the nucleotide alignment similarity and substitution mutations in all of cysteine protease, amoebapore and Gal/GalNAc lectin. Phylogenetic tree analysis based on cysteine protease gene partial sequence in local E.histolytica human isolates that used for genetic variation analysis showed genetic closed related to NCBI-BLAST E.histolytica Japan strain (AK421547.1) at total genetic changes (0.0060-0.0010%), while Phylogenetic tree analysis based on amoebapore B gene partial sequence in local E.histolytica human isolates that used for genetic variation analysis showed genetic closed related to NCBI-BLAST E.histolytica, Germany strain (X76904.1) at total genetic changes (0.0060-0.0010%). Phylogenetic tree analysis based on Gal/GalNAc lectin gene partial sequence in local E. histolytica human isolates that used for genetic variation analysis showed genetic closed related to NCBI-BLAST E.histolytica Bangladesh strain (AF501280.1) at total genetic changes (0.0060-0.0020.

Analysis of Cathepsin-L and Cathepsin-B Gene Expression in Embryonated Eggs of Fasciola gigantica and Effect of Various Protease Inhibitors on Miracidial Hatching

Background: Fasciola gigantica is a helminth parasite of veterinary importance and a constraint on the growth and productivity of domestic livestock in the tropical countries of Asia and Africa. Present study aimed at determining the expression of four cysteine protease genes including cathepsin (cat) L-1 D and cat-Bs in the embryonated egg stage of F. gigantica by polymerase chain reaction (PCR). In addition to the analysis of expression of the above genes in the embryonated eggs, inhibition of in vitro hatching of the miracidia from the eggs was carried out using three protease inhibitors viz. serine, cysteine and leucine aminopeptidase inhibitors to understand the possible role of these proteases in the hatching of the miracidia from the eggs. Methods: Cat- L-1D, cat B-1, cat B-2 and cat B-3 genes were amplified by reverse transcriptase and quantitative-PCR, respectively for determining their expression in the embryonated eggs. Inhibition of in vitro hatching of the miracidia from the eggs was carried out by exposing the embryonated eggs to different concentrations of iodoacetamide, phenyl methyl sulphonyl fluoride and bestatin as cysteine, serine and leucine aminopeptidase inhibitors. Result: Results showed that cat L1-D, cat B-1 and cat B-3 genes were expressed at the embryonated egg stage but cat B-2 gene was not expressed at this stage of the parasite. Iodoacetamide and phenyl methyl sulphonyl fluoride (cysteine and serine protease inhibitors) inhibited the miracidial hatching at different concentrations, indicating probable role of cysteine and serine proteases in its hatching process. However, leucine aminopeptoidase does not seem to influence the miracidial hatching from the eggs as bestatin did not inhibit its hatching.

A new cysteine protease allergen from Ambrosia trifida pollen: proforms and mature forms

Giant ragweed (Ambrosia trifida) pollen is closely associated with respiratory allergy in late summer and autumn, and the prevalence of giant ragweed pollen allergy progressively increases. Compared with short ragweed (Ambrosia artemisiifolia), allergenic components from giant ragweed pollen are poorly investigated. To promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, it becomes necessary to identify and characterize unknown allergens from giant ragweed pollen. In the present study, we identified and characterized a new cysteine-protease (CP) allergen from giant ragweed pollen, named as Amb t CP. The cloned Amb t CP gene encoded 387 amino acids. Recombinant Amb t CP (rAmb t CP) and natural Amb t CP (nAmb t CP) were purified by high-affinity Ni2+ resin and immunoaffinity chromatography respectively. During refolding, purified rAmb t CP could autocatalytically converted to its mature forms displaying a higher enzymatic activity. Moreover, the autocatalytic conversion of proforms to mature forms of nAmb t CP could cause their amount to change in giant ragweed pollen extracts. Then, the allergenicity of Amb t CP was characterized: 23 (33.8%) of 68 Chinese patients with ragweed pollen allergy showed positive IgE binding to nAmb t CP by enzyme-linked immunosorbent assay (ELISA); the result of subsequent ELISA showed that IgE-binding activity of proforms and mature forms of rAmb t CP was different, with positive rate of 39.1% (9/23) and 47.8% (11/23) respectively; Amb t CP showed IgE cross-reactivity with the CP components from short ragweed, Artemisia annua and Artemisia sieversiana pollen. Our findings will help to promote component-resolved diagnosis and treatment for giant ragweed pollen allergy, standardize allergen products and individualize allergen-specific immunotherapy.

Recombinant cysteine proteinase as anti-tick targeting Hyalomma asiaticum infestation

Cysteine proteases are involved in the digestion of host blood and the degradation of yolk proteins of arthropod ectoparasites. In this study, a cathepsin L-like cysteine proteinase gene (HasCPL) of Hyalomma asiaticum was cloned, and recombinant (r)HasCPL protein was generated for immunization study. Bioinformatic analysis confirmed HasCPL was a member of the papain family (clan CA) and have high sequence identities with CPLs of other Ixodid ticks. The efficacy of immunization against H. asiaticum infestations in rabbits was assessed. Rabbits (n = 3) were immunized three times with rHasCPL before challenged with 250 larvae per rabbit four weeks post-immunization. A high antibody titer was detected in immunized rabbits in comparison to control. Western blot analysis detected CPLs in midgut, salivary gland, and ovary. Increase of rejection percentage of larvae were noted in ticks fed on immunized animals in comparison to control. Overall, a 55.09% protection against larva ticks was noted.

Heterologous expression of the cysteine proteinase gene VaCP17 from the cold-adapted grapevine Vitis amurensis increases cold tolerance in Arabidopsis

Cysteine proteinases (thiol) carry out diverse and critical functions in plants through their ability to hydrolyze peptide bonds in target proteins. Here, we cloned a cysteine proteinase gene designated VaCP17 from a highly cold-resistant wild Vitis amurensis accession ‘Shuangyou’, and then its potential function in cold resistance was investigated. The results showed that the CDS of VaCP17 is 1404 bp, encoding 467 amino acids, the VaCP17 protein localized to the cell membrane. Expression of CP17 was highly distinctive among different structures of ‘Shuangyou’ and the cold-sensitive Vitis vinifera cultivar ‘Red Globe’, with the highest expression in the stem of ‘Shuangyou’ and the flower of ‘Red Globe’. Arabidopsis plants constitutively expressing a VaCP17-GREEN FLUORESCENT PROTEIN fusion (35S::VaCP17-GFP) showed increased survival after transient exposure to freezing (-6 °C), and showed lower electrolyte leakage and MDA content, higher soluble sugar content and SOD, POD and CAT activities, as compared with non-transgenic Arabidopsis controls. The expression of nine cold-resistance related genes (CBF1, CBF2, CBF3, RD29A, COR15A, KIN1, NCED3, AOC1 and JAZ10) in 35S::VaCP17-GFP plants was increased under cold treatment at 4 °C, relative to control plants. Using a yeast two-hybrid system, we identified VaNAC72, VaCAM7 and VaDi19 as potential interactors of VaCP17, and their interactions were demonstrated by a bimolecular fluorescence complementation assay. In conclusion, we revealed that VaCP17 can enhance cold resistance by influencing physiology and biochemistry and the expression of cold resistance related genes under cold stress.

How Cysteine Protease Gene PtCP5 Affects Seed Germination by Mobilizing Storage Proteins in Populus trichocarpa.

In higher plants, seed storage proteins are deposited in protein storage vacuoles (PSVs) and degraded by protease, especially cysteine proteases, as a source of nitrogen for seed germination. In this study, a cathepsin B-like cysteine protease PtCP5, which is important for seed germination and pollen development, was first cloned in Populus trichocarpa. The GUS staining of the ProPtCP5-GUS reporter line showed that PtCP5 is expressed in the roots, stems, leaves, flowers, siliques and seeds of Arabidopsis. We reveal that PtCP5 is present in plasma membrane and co-localizes with the plasma membrane marker REM1.3. Both seed germination and early seedling development are slower in OX-PtCP5 transgenic Arabidopsis when compared with the wild-type. Further analysis revealed that, when stained with toluidine blue, the observed storage protein accumulation was lower in OX-PtCP5 than in the wild-type. Our results also show that the number of abnormal pollen grains is higher and the germination rate of pollen is lower in OX-PtCP5 than in the wild-type. These results indicate that PtCP5 is an important factor in mobilizing storage proteins and that the proper expression of PtCP5 is necessary for both pollen and seed maturation and germination. This study sheds further light on the biological functions of cysteine proteases and provides further reference for seed development research on woody plants.