Expression And Function Analysis Of Seed Hardness-related Genes PgL0145810.1 And PgL0007350.1 In Pomegranate (Punica Granatum L.)

Pomegranate(Punica granatum L.)is native to central Asia,which is an important fruit contributing to poverty alleviation and rural revitalization.Fruit traits in pomegranates are the basic criteria for the consumers in selecting quality fruits.Some of the major traits representing the quality of the products are fruit color,seed hardness,aril color,taste,etc.The edibility of the fruits of pomegranate depends on the fleshy outer seed coats and hard or soft inner seed coat.It is the key factor for the preferences by the consumers to particular varieties.The consumers' preference towards soft-seeded pomegranates has accelerated the breeding to new soft-seeded pomegranate cultivars.However,the seed coat development in pomegranate has not yet been defined.Seed hardness is an important trait in pomegranate that determines the edibility and quality of the fruit.It is a major factor for the economic value of the fruit as soft seeded fruits fetch a higher price in the market compared to the hard seeded ones.Although some research works on seed hardness traits in pomegranate,there are no clear molecular mechanisms and identification of genes responsible for seed hardness.In our experiment,we researched the genetic mechanism of seed hardness trait in pomegranate by identifying the differentially expressed genes among the soft seed and hard seeded varieties and further studied the expression of those genes through cloning in the Arabidopsis plants.For this study,we considered seeds of three soft seeded and three hard seeded pomegranate varieties from the nursery of Zhengzhou Fruit Research Institute,Henan.The outcomes of our research are as follows:1.Sucrose,a critical sugar,is transported from source to sink tissues through the phloem and plays a vital role in developing important traits in plants.However,the SUT gene family is still not well characterized in pomegranate.In this study,we first identified the pomegranate sucrose transporter(SUT)gene family from the whole genome sequence of pomegranate 'Tunisia.' We identified a total of ten SUT genes which were divided into three categories.The phylogenetic relationship of SUT gene structure and their promoters were analyzed.Their expression patterns were detected during the seed development.The average GC content in the pomegranate SUT gene family members is 44.35% which is lower than the average GC content of Arabidopsis and rice SUT genes family.At the same time,the average length of the pomegranate SUT genes is(4406.7bp),which is higher than that of Arabidopsis(1538.7bp)and rice(1608.6bp).The promoter region of the pomegranate SUT genes contained more than twenty MYB elements,where nine of them are major ones.2.Four of the SUT genes,Pg L0328370.1,Pg L0099690.1,Pg L0145810.1,and Pg L0145770.1,were differentially expressed during the seed development.Pg L0099690.1 down-regulated at 120 days after flowering in ‘Sanbai' seeds than at 60 days after flowering.The Pg L0145810.1 and Pg L0145770.1 both down-regulated in ‘Tunisia' seeds at 120 days after flowering compared to that at60 days after flowering.Conversely,Pg L0328370.1 up-regulated at 120 days after flowering in ‘Sanbai'or ‘Tunisia' seeds than at 60 days after flowering.For the different cultivars,Pg L0145770.1down-regulated at 60 days after flowering in ‘Tunisia' seeds compared to that in ‘Sanbai.'3.We found that Pg L0145810.1 gene expressed differentially among the hard seeded and soft seeded pomegranate varieties from the transcriptomic data analysis.To further investigate the function of Pg L0145810.1,we cloned Pg L0145810.1 and dipped the flower buds of Arabidopsis in an Agrobacterium cell suspension.The transgenic Arabidopsis lines were confirmed through q RT-PCR analysis.The transgenic lines were grown for three generations(T3)to obtain homozygous plants for the transferred gene.Growth phenotypes of the Pg L0145810.1 transgenic line were highly different from the wild type in terms of plant height,number of leaves,number of stems,and seeds per siliques.We further noticed that Pg L0145810.1 was expressed most prominently in the stem parts in transgenic plants compared to other tissue parts(leaves,flowers,and silique).To further assess the function of Pg L0145810.1,the cytological observation was conducted in the tissue of stems from the thirty-five-day-old seedling stage collected from WT,L4,and L12 plants.The cells in the xylem vessels were small,and lignin content was lower in the transgenic plants than wild Arabidopsis plants.Similarly,the expression patterns of Pg L0145810.1 in the stems of L4 and L12 were higher.In general,our result suggests that the MYB cis-elements in the promoter region might regulate Pg L0145810.1expression to control the structure of the xylem,thereby affecting seed hardness in pomegranate.4.From the transcriptomic data,we found many genes regulating and controlling the seed hardness trait in pomegranate.We considered a WRKY TF gene Pg L0007350.1 which expressed differentially in hard and soft seeded pomegranate varieties for our further experiment.Thus,to better understand the function of the gene,we performed cloning of Pg L0007350.1 into the Arabidopsis plant.We considered over-expressed transgenic lines to the Pg L0007350.1 gene and found that the transgenic lines showed increased biomass density and thicker cell wall in the vascular bundle cells wild-type(WT)plants.Besides,the transgenic lines had higher contents of lignin in the inflorescence stem.Our findings support that the Pg L0007350.1 gene act in cell wall regeneration,secondary cell wall formation,and xylem lignification,which is correlated to the seed development in the pomegranate fruit.An increase in the lignin content in the cortex of the cell might have caused hardness in the seeds of the pomegranate.Thus,we consider the regulation of the Pg L0007350.1 gene to produce a soft seeded variety of pomegranate through genetic engineering.