Progresses in research on pomegranate seed hardness

Abstract: Pomegranate (Punica granatum L.) is a fruit species cultivated worldwide. Pomegranate fruits possess rich nutrition, unique flavor and important economic value, and it is favorite by people for its nutrients benefiting to human health. As more attentions are being paid to the nutrition and health protection functions of pomegranate fruits, its market demand has increased rapidly and the industry has developed gradually. Pomegranate cultivars can be divided into soft- seeded cultivars, semi- soft-seeded cultivars, hard-seeded cultivars, and so on. Seed hardness is one of the important quality charac-teristics of fresh pomegranate, which determines the sensory quality of the fruit and consumer accep-tance. In recent years, with the increasing attentions on the seed hardness of pomegranate fruits, there were many studies focusing on the seed hardness formation and soft-seed pomegranate breeding, so as to understand the reason for pomegranate seed hardness, and provide a reference to further study the ge-netic mechanism of seed hardness formation in pomegranate. In the present review, seed hardness for-mation and its influencing factors are reviewed, and candidate genes involving in seed hardness forma-tion and the heredity patterns of seed hardness are summarized. As we known, there are two layers of seed coats for pomegranate, that is, outer and inner seed coats. The soft and juicy outer seed coat is rich in nutrients, which is the main edible part of pomegranate fruits. The inner seed coat is rich in lignin and cellulose, which forms the seed hardness. The seed hardness formation is a process of lignin accu-mulation in endoderm cells and thickening of secondary cell walls, namely, a process of lignification of endoderm cell walls. The seed hardness is depended on cultivars, growing technology and environment, while the genetics of cultivars plays the decisive roles. Lignin is composed of the primary monolignols  p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, which form predominantly by oxidative po-lymerization of the three major monolignols to generate the hydroxyphenyl (H), guaiacyl (G), and syrin-gyl (S) lignin subunits. Nontarget metabolic profiling revealed that coniferyl alcohol and sinapyl alco-hol were main monolignols accumulated in seed coat of pomegranate, while little p-coumaryl alcohol was detected in the inner seed coat. Thus, S lignin and G lignin appeared to be the dominant lignin sub- units in pomegranate seed coats. In addition, the accumulation of cellulose and hemicellulose in seed coat was significantly different between hard-seed and soft-seed pomegranates, which suggested that the accumulation difference of cellulose and hemicellulose in seed coat was also an important factor in the formation of pomegranate seed hardness. Several genes involving in lignin, cellulose and hemicellu-lose synthesis and degradation had special expression during seed hardness formation, which were iden-tified and considered as the potential candidate genes involving in seed hardness formation. Pgr011171.1 encoding POD, Pgr013634.1 encoding F5H, Pgr022328.1, Pgr006310.1, Pgr006318.1, and Pgr006319.1 encoding CAD, and Pgr011171.1 encoding POD had lots of transcript accumulation, accompanied with the sinapyl alcohol and coniferyl alcohol accumulation, were candidate genes involv-ing in seed hardness formation. Basing on the results of co-expression network analysis of lignin, cellu-lose and hemicellulose metabolism-related genes and the transcription factors for inner seed coats at dif-ferent developmental stages with both hard-seed cultivar and soft-seed cultivar, Pgr000815.1 encoding AUX-IAA might be involved in the lignin metabolism, and Pgr011491.1 and Pgr022940.1 encoding AUX-IAA2, and Pgr017424.1 encoding NAC66 might be involved in the metabolism of cellulose and hemicellulose. PgL0137670 encoding NAC transcript factor has a SNP site (T-C) at 166 bp, and the al-lelic variant T site of PgL0137670 is associated with the characteristics of soft-seed, according to the comparative transcriptome analysis of soft-seeded cultivar and hard-seeded cultivar. Besides, a number of selective sites related to seed hardness including SUC8, SUC6, FOXO and MAPK genes were select-ed basing on the whole genome resequencing analysis with different seed hardness varieties. However,the gene functions need further studies. The growth environment also has a certain impact on the seed hardness of pomegranate. Analysis of the seed hardness of pomegranate grown in different soils found that the seed hardness of pomegranate grown in sandy soil was the highest, followed by loam soil and the smallest in red loam soil. The study on the effect of different fertilization treatments on the seed hardness of pomegranate found that the application of phosphorus fertilizer could reduce the seed hard-ness, while the application of fertilizer such as urea, compound fertilizer and potash fertilizer had no sig-nificant effect on the seed hardness. Gibberellin, 2,4-dichlorophenoxyacetic acid (2,4-D) and naphtha-lene acetic acid are widely used plant growth regulators, and they have significant effects on improving fruit quality. Research showed that foliar spraying of these growth regulators during pomegranate flow-ering could significantly reduce the seed hardness of pomegranate. Although the study of hormones in regulating the seed hardness of pomegranate has been reported in related studies, in the actual produc-tion process, there is still a lack of suitable plant growth regulator application technology to promote pomegranate seed softening. In conclusion, seed hardness is one of the characteristics of pomegranate. Although different cultivation environments or artificial measures have a certain impact on the seed hardness of pomegranate, this effect is relatively limited, and genetics is the fundamental factor that determines seed hardness. Finally, the new pomegranate cultivars, bred independently in China and having soft or semi-soft seeds, are introduced in the review. Summarily, the review will provide not only the insight to further study on mechanism and regulation of seed hardness formation, but also the guid-ance for genetic improvement and breeding in pomegranate.