Vicilin and legumin storage proteins are abundant in water and alkali soluble protein fractions of glandless cottonseed

www.nature.com/scientificreports OPEN Vicilin and legumin storage proteins are abundant in water and alkali soluble protein fractions of glandless cottonseed Zhongqi He1*, Christopher P. Mattison1, Dunhua Zhang2 & Casey C. Grimm1 In this work, we sequentially extracted water (CSPw)- and alkali (CSPa)-soluble protein fractions from glandless cottonseed. SDS-Gel electrophoresis separated CSPw and CSPa to 8 and 14 dominant polypeptide bands (110–10 kDa), respectively. Liquid chromatography-electrospray ionizationtandem mass spectrometry identified peptide fragments from 336 proteins. While the majority of peptides were identified as belonging to vicilin and legumin storage proteins, peptides from other functional and uncharacterized proteins were also detected. Based on the types (unique peptide count) and relative abundance (normalized total ion current) of the polypeptides detected by mass spectrometry, we found lower levels (abundance) and types of legumin isoforms, but higher levels and more fragments of vicilin-like antimicrobial peptides in glandless samples, compared to glanded samples. Differences in peptide fragment patterns of 2S albumin and oleosin were also observed between glandless and glanded protein samples. These differences might be due to the higher extraction recovery of proteins from glandless cottonseed as proteins from glanded cottonseed tend to be associated with gossypol, reducing extraction efficiency. This work enriches the fundamental knowledge of glandless cottonseed protein composition. For practical considerations, this peptide information will be helpful to allow better understanding of the functional and physicochemical properties of glandless cottonseed protein, and improving the potential for food or feed applications. With about 28 × 109 kg or 124 million bales of cotton produced annually, cotton plant (Gossypium hirsutum L.) is an economically important crop known for fiber, cottonseed oil, and protein contents1–3. Currently, cotton fiber and cottonseed account for 85–90% and 10–15% of the value of the crop, respectively, while 150 kg of cottonseed is produced for every 100 kg fiber ginned. The cottonseed consists of approximately 16% oil, 45% meal, 25% hull, and 8% linters. The major components of defatted meal are carbohydrates and protein. Cottonseed protein comprises multiple polypeptides (i.e., various protein fractions) including seed storage proteins and proteins with other biological f unctions4,5. Cottonseed protein has great potential as a component of value-added industrial products and bio-active functional materials. The potential added-value products of cottonseed protein isolates include, but are not limited to, bio-based a dhesives6–10, bioplastics and fi lms11,12, antioxidant fractions/ 13,14 15 peptides , and antibiotic p eptides . The traditional variety of cottonseed contains the toxic terpenoid gossypol and is labeled “glanded cottonseed” as gossypol is deposited in scattered tissue structures called glands. Research efforts have been made to produce a new type of “glandless” cottonseed in which there is only trace gossypol content. Gossypol is a terpenoid produced and stored in the pigment glands of the typical cotton plant (about 1 weight percent (wt%) of dry matter of cottonseed)16. Thus, the traditional variety of cottonseed is also labeled as “glanded” c ottonseed17,18. The presence of toxic gossypol limits cottonseed to mainly non-food applications1. Research has been directed towards eliminating or reducing gossypol from cottonseed (i. e., below 450 ppm free gossypol) to mitigate the toxic effects of gossypol. One notable genetically modified cultivar, TAM66274, has ultra-low gossypol cottonseed16. Another interesting genetically engineered cultivar is NuMex COT 15 GLS’ Glandless C otton17. Cheng et al.19,20 demonstrated that the glandless cottonseed protein isolates could serve as wood adhesives. He et al.21 reported that glandless cottonseed protein-based adhesives had similar bonding performances as the glanded counterpart. In the latest study, He et al.14 comparatively examined the antioxidant activities of the water-soluble fractions of 1 USDA-ARS, Southern Regional Research Center, New Orleans, LA 70124, USA. 2USDA-ARS, Aquatic Animal Health Research Unit, Auburn, AL 36832, USA. *email: Scientific Reports | (2021) 11:9209 | https://doi.org/10.1038/s41598-021-88527-7 1 Vol.:(0123456789) www.nature.com/scientificreports/ Figure 1.  Gradient (10–20% Tricine) SDS-PAGE of water- (CSPw) and alkali- (CSPa) soluble cottonseed protein isolates with ( +) or without (−) dithiothreitol (DTT) treatment. Approximately 2 µg of protein were applied to each lane. Gels were visualized and protein band intensities were quantified using the 680 nm channel on a Li-Cor Odyssey infrared scanner and Image Studio software (LI-COR Biosciences, USA). The SDS-PAGE profile of corresponding glanded samples can be found in He et al.4. both glandless and glanded cottonseed protein. Cao et al.18,22 reported ethanol extracts from both glanded and glandless cottonseed kernels contained some bioactive ingredients. Continued characterization of glandless cottonseed will enable improved understanding and food and feed utilization of glandless cottonseed protein. In this work, we isolated and separated glandless cottonseed proteins into water- and alkali-soluble fractions, analyzed their polypeptide profiles, and compared them to the peptide features of glanded cottonseed protein fractions. Results and discussion Polypeptide band features of glandless CSPw and CSPa on gradient SDS‑PAGE. SDS-gel electrophoresis separated CSPw and CSPa into 8 and 14 discernable polypeptide bands, respectively (Fig. 1, fulllength gels and blots are included in a Supplementary Information file). The polypeptide bands ranged from over 100 to about 11 kDa with CSPa, but just over 50 to 10 kDa for CSPw. Among the 14 CSPa bands, Ca 5 and Ca 6 at 55 and 45 kDa were most abundant. Together, they accounted for 43% of the relative abundance of the total protein load (Fig. 2). In contrast, the three most abundant bands in the CSPw sample (Cw 5, 6 and 7) were about 20, 15 and 13 kDa. Together, they accounted for 45% of the relative abundance of the total CSPw protein load. These observations were generally consistent with previous data of glandless cottonseed p roteins14,23. Similar protein band patterns with minor differences were observed when comparing glandless to glanded protein extract. There were more CSPa bands, but less CSPw bands in the glandless sample when compared to a glanded sample with 7 CSPa and 12 CSPw b ands4. The main difference in the glandless CSPa bands was the four molecular weight bands from 60 to 110 kDa (Ca 1 to 4) which were not observed in either the glanded CSPa fraction4,24 or in glanded whole cottonseed protein isolates5,24,25. The presence of gossypol could result in modified SDS-PAGE protein patterns due to the chemi (...truncated)