Analysis of cellulose content and synthase gene expression in juice sacs secondary wall during granulation of Guanxi pomelo

Abstract:【Objective】Juice sacs granulation often occurs during the period of ripening and storage in pomelo. It is mainly caused by thickening of secondary walls, resulting in the loss of flavor, and edible value. There have been many researches on the causes of the granulation, but the regulation mechanism is still unclear. Therefore, it is necessary to study the correlation between the granulation and the second- ary wall cellulose content, free water and bound water content, quality and other indicators, as well as the expression of cellulose synthase genes in the juice sacs of pomelo.【Methods】The fruits of Guanxi pomelo in the four periods of 8 (immature), 9 (initial maturity), 10 (maturity) and November (hanging on the tree for a month) were selected as the experimental materials. Firstly, the fruit shape index and the granulation rate of the juice sacs were counted. Total soluble solid (TSS) was determined by a hand-held refractometer. The content of titratable acid (TA) was determined by acid-base neutralization titration. The free water and bound water content of the juice sacs was measured by sucrose solution soaking method. The content of cellulose was determined by anthrone method. A total of 18 biological repeats were set for monthly samples, and each biological repeat was set with 3 technical repeats. The paraffin  sections were stained with safflower solid green dye and examined with LEICA DMI 48 inverted fluores- cence microscope. SPSS 22.0 was used for correlation analysis. Secondly, the expression of cellulose syn- thase genes (CESA) was identified through the four transcriptomes, and the heat map analysis of the expression volume was carried out with TBTools software to find the differentially expressed CES A genes. Thirdly, MEGA 6.0 software was used to analyze the evolutionary tree of these differently-expressed CE- SA genes with the related CESA genes in Arabidopsis thaliana, Oryza satvia, Populus tremuloides, Zea mays and Gossyplum hirsutum, and predict their function. Finally, RNA was extracted using the univer- sal RNA extraction kit RT411 (Skyroot Bio). cDNA was transcribed using Prime Script TM RT Reagent Kit with gDNA Eraser RNA Reverse Transcription Kit (TaKaRa). SYBR Green I Kit (TaKaRa) was used for real-time fluorescence quantitative PCR amplification, by which The expression level of the CESAgenes in the four stages of the juice sacs were analyzed.【Results】When the fruits of Guanxi pomelo en- tered the mature stage (September and October), the juice sacs were softened and juicy, the TSS content was high, the TA content was low, the flavor was good, and it was the best time for eating, although some fruits began granulation slightly. After October, the transverse diameter of the fruits increased rap- idly, the granulation of the fruits accelerated significantly, and the granulation of single juice sac was se- vere. The granulation rate increased from 0% to 11%. The content of cellulose, TA and bound water con- tent increased significantly, while the fruit shape index and free water content were reduced significant- ly, which greatly affected the taste of the fruits; Using phylogenetic tree analysis, five differentially expressed genes CrCESA1-1, CrCESA2-2, CrCESA4, CrCESA7-1 and CrCESA8 were screened out by transcriptomic analysis. The CrCESA4, CrCESA7-1 and CrCESA8 were found to be clustered with Ara- bidopsis AtCESA4, Uland cotton GhCESA7 and Arabidopsis AtCESA8, respectively. With the exception of the CrCESA1-1, the relative expression of other genes was consistent with the transcriptome, account- ing for 80%. The CrCESA2-2 was highly expressed in August and November, and the CrCESA4, CrCE- SA7-1 and CrCESA8 increased significantly in transcriptional and expression levels in November.【Conclusion】During the mature stage of Guanxi pomelo fruits in September and October, the degree of the juice sacs granulation was closely related to the increase of secondary wall cellulose content, the de- crease of fruit shape index and free water content, the increase of bound water and TA content. The rCE- SA4, CrCESA7-1 and CrCESA8 might be involved in the process of secondary wall thickening.