Safflower (Carthamus tinctorius L.), belongs to Compositae, is an annual or biennial herb, also known as safflower, safflower or sub-safflower and so on. It is found that up to now, more than 200 compounds have been isolated from Carthamus tinctorius, among which more than 104 compounds have been identified. these compounds are: flavonoids, fatty acids, alkaloids, polyacetylene, spermidine, lignans, sesquiterpenes, sterols, polysaccharides and other components. Modern pharmacological experiments also show that Carthamus tinctorius and its active compounds have a wide range of biological activities, which can dilate coronary arteries and improve myocardial ischemia. Regulate the immune system, anticoagulation and antithrombotic, antioxidant, hypoxia tolerance, anti-fatigue, anti-liver fibrosis, anti-tumor, anti-inflammation, analgesia and so on [3, 4]. Because safflower seed oil is rich in unsaturated fatty acids, it can be used as edible oil, especially linoleic acid, with a content of about 80% in safflower seeds, which is called "king of linoleic acid" . Therefore, in many countries, such as India, Mexico, the United States, Ethiopia, Spain and Australia, safflower seed oil has become a common plant edible oil .
Cysteine protease (EC3.4.22), also known as mercaptan protease, is a large class of proteolytic enzymes. The international classification of proteases is based on the active center of the enzyme. The active site of cysteine protease contains nucleophilic Cys residues, which together with the other two amino acids form Cys-His-Asn/Glu catalytic triad to catalyze the hydrolysis of corresponding proteins[6, 7]. Cysteine protease exists in animals, plants and microorganisms, and is widely involved in a variety of life activities. Plant cysteine proteases are divided into 5 families (CA, CD, CE, CF, PC) and 15 families.With the sequencing of the genomes of a large number of species, cysteine proteases in plant genomes have been reported in Arabidopsis, citrus, papaya, rubber and other dicotyledonous plants and rice. Cysteine proteases are very conservative among different species. in the process of evolution, cysteine proteases are divided into several subfamilies. As early as 2004, Beers and his colleagues identified and classified 138 cysteine proteases in plants. According to the clustering characteristics, these proteases were divided into 8 subfamilies . Richau et al divided 723 species of cysteine proteases into 9 subfamilies in 2012. The results of the two classifications were similar. The latter subdivided the subfamily RD21 into subfamilies RD21 and XBCP3  according to sequence characteristics. At present, the cysteine protease gene family is generally divided into 9 subfamilies, named RD21, CEP, XCP, XBCP3, THI, SAG12, RD19, ALP and CTB9 respectively. Its Central Asian family RD21, CEP, XCP, XBCP3, THI, SAG12 is named as the cath L-like subfamily, while the subfamilies RD19, ALP and CTB9 are named as the cath Fmurike subfamily cath Hliklippe cath B-like subfamily. The subfamily cath Lmurike in plants, cathepsin L, F, H, B, correspond to the subfamilies cathepsin L, F, H, B  in animals, respectively.
Similar to caspases, which regulates apoptosis in animals, cysteine proteases in plant cells are involved in programmed cell death (programmed cell death, PCD) [11, 12]. Arabidopsis thaliana XCP1 and XCP2 are specifically expressed in xylem and control catheter programmed death . In the process of pollen development, the tapetum needs to be degraded during pollen maturation, and the tapetum needs to undergo programmed death to release nutrients and ensure the synthesis of the pollen wall. CEP1 is a cysteine protease involved in programmed tapetum cell death and pollen development. Cep1 mutant tapetum cells can not be degraded, pollen fertility decreased, pollen exine abnormal . Song Liping cloned two cysteine proteases Bna C.CP20.1 and Bna C. CP13.4 in Brassica napus and used tapetum-specific promoters to initiate the expression of these two genes. Transgenic plants did not pollinate due to premature tapetum degradation at tetrad stage. Therefore, it is very important for cysteine protease to participate in tapetum PCD during tapetum degradation during pollen development. In tobacco, programmed cell death of embryo sac stalk is regulated by CP14 and its suppressor gene CYS. Knockout of CP14 leads to delay of PCD of embryo sac stalk, while inhibition of CYS expression leads to advance of  embryo sac stalk PCD. It is reported that tobacco Cathepsin B is necessary for allergic reaction and resistance caused by non-host pathogens [16–18]. Arabidopsis ctb mutants can reduce programmed cell death induced by biological stress . Further studies have shown that silencing Cathepsin B reduces ROS accumulation and endoplasmic reticulum stress-induced programmed death (ER-stress-induced PCD, ERSID).
Plant senescence is a physiological phenomenon caused by internal and environmental factors. Senescence will be accompanied by the degradation of chlorophyll, the decline of photosynthesis, the degradation of macromolecules, the redistribution of nutrients, and the degradation of cells until the final death. Protein degradation, especially the degradation of target proteins in chloroplasts, is an important hydrolysis process in plant leaf senescence. A member of the SAG12,cath L-like subfamily in Arabidopsis thaliana is currently a marker gene for leaf senescence, . Under normal conditions, the phenotype of sag12 mutant did not change significantly, but the yield of sag12 mutant decreased under low nitrogen condition, indicating that sag12 was involved in the reuse of nitrogen in seeds . SAG12 and RD21A in rape were reported to be related to protein degradation and nitrogen limitation in senescent leaves [23, 24]. There are 17 Arabidopsis SAG12 homologous genes  in rubber, in which Hb SAG12H1 is used as a molecular marker of senescence. Six genes Hb RD21B, Hb RD21E, Hb RD21F, Hb CEP1, Hb XBCP3L, Hb RD19B which play an important role in leaf senescence were identified by deep sequencing in papaya. In Arabidopsis thaliana, XCP1 and XCP2 are associated with senescence, and XCP1 is specifically expressed in xylem, which leads to a decrease in chlorophyll when it is ectopic in leaves. Barley is a model plant for the study of leaf senescence in monocotyledons. Hv Pap-1 accumulates  in senescent flag leaves and primary leaves induced by darkness. Van Wyk et al identified a large number of papaya cysteine protease  during the senescence of soybean nodules. Eggplant SmCP expressed  in senescent leaves, fruits, xylem and anthers.
Plants always encounter abiotic stress in the process of growth, and the recovery and utilization of protein through protein hydrolysis is the main line of defense for plant survival. A large number of studies have shown that cysteine protease up-regulates the expression of cysteine proteases in plants under environmental stress. AtRD21A and AtRD19A are markers of dehydration response and up-regulate the expression of  after drought and salt stress. Under PEG, salt and cold stress, TaCP expression in wheat was up-regulated by . Arabidopsis thaliana overexpressing Ta CP was more resistant to drought than wild type under drought stress, suggesting that TaCP plays an important role in response to drought. Sweet potato SPCP2 is expressed in naturally senescent leaves, and darkness, ABA, JA and ethephon can also induce the expression of SPCP2 . Arabidopsis thaliana transformed with SPCP2 is more resistant to salt and drought than wild type . Sweet pepper CaCP expression was induced in senescent leaves, and was significantly up-regulated under biotic and abiotic stress, which inhibited CaCP expression and enhanced plant resistance to salt and osmotic stress .
Based on the data mining and processing of the whole genome of Carthamus tinctorius, 25 cysteine proteases were identified in Carthamus tinctorius. According to the evolutionary relationship, these members were divided into 9 subfamilies. We further analyzed the basic properties of the protein, the conserved domain, the gene family structure, the cis-acting element on the promoter, the expression pattern of the gene at different flowering stages and the changes of gene expression under stress, and cloned a gene CtCP1 that responded to low temperature. Inhibition of CtCP1 gene expression enhanced the resistance of transgenic Arabidopsis to low temperature stress, while overexpression of CtCP1 gene reduced the resistance of transgenic Arabidopsis thaliana to low temperature stress. As a hydrolase, cysteine protease may play a broad-spectrum resistance role in plant abiotic stress. In this study, the use of genome-wide biological analysis and transgenic materials are helpful to analyze the molecular mechanism of the effect of cysteine protease on plant freezing resistance, and provide theoretical guidance and genetic resources for the breeding of freeze-resistant safflower varieties.