Mechanism Study On The Lignin Accumulation In Lignified Cells And Cell-wall Dynamic Of Pectin In Postharvest Loquat Fruit

Loquat is a kind of characteristic seasonal fruit in China,which tastes soft and juicy,and is popular among consumers.Loquat fruits are harvested in the high temperature and humidity summer season.The quality of loquat is rapidly degraded at ambient temperature after harvest.Low temperature storage is an important approach to delay quality decrease of loquat fruit.However,red-flesh loquat fruit undergo chilling-induced lignification when stored at a temperature below 5°C,exhibiting texture deterioration such as increased firmness and increased lignin,which result in a rapid decline in the commercial value of the fruit.The postharvest lignification become one of the bottlenecks restricting the development of the loquat industry.At present,the research on postharvest lignification of loquat fruit mainly focuses on the analysis of the physiological,biochemical and molecular biological mechanisms of lignin accumulation.There are few studies on the specific accumulation of lignin at the single-cell level of loquat fruit and the homologous transient expression of related genes in loquat cells.At the same time,there is a lack of research on the non-lignin compositions in the cell wall of the postharvest loquat fruit,especially the changes in the cell level of the pectin composition and its cell-wall dynamics mechanism.In this study,red-flesh loquat fruit‘Luoyangqing’were used,which were stored at 0°C and low temperature conditioning,separately,to obtain loquat fruit with different degrees of lignification.Methods including label-free molecular spectroscopic imaging,transient transformation of loquat cells,and fluorescent protein fusion were used to analyzed the development mechanism of lignified cells during postharvest lignification of loquat fruit,transient expression study of key lignin coding genes and transcription factors,investigate the transient transformation of lignin synthesis-related coding genes and transcription factors in loquat cell and reveal the cellular biological mechanism of lignin accumulation during postharvest lignification of loquat fruit;At the same time,based on methods such as immunofluorescence histochemistry,immunofluorescence intensity quantification,oligogalacturonide probe（OG）labeling,fruit injection in situ,etc.,the key pectin composition and structural domains that cause the deterioration of loquat texture were screened and verified in situ,and the cell-wall mechanism of the pectin was analyzed at the level of cell biology during postharvest lignification of the loquat fruit.1.A development mechanism of lignified cells in loquat fruit was proposed,and the accumulation mechanism of cell-wall compositionts during the development of lignified cells was studied at the signle-cell level.The distribution of lignin on the equatorial surface of the loquat flesh was obtained at the tissue level.The density of lignified cells was significantly related to the lignin staining area ratios,lignin content,and fruit firmeness.It is speculated that the increase in the number of lignified cells may be an important factor in the postharvest lignification of loquat fruit.The simultaneous in situ imaging of lignin,cellulose and pectin in different morphological lignified cells was realized at the cell level,and the development mechanism of lignified cells was proposed.It is believed that the accumulation of lignin and cellulose in lignified cells is the main driving force for its development.This study also found that a few parenchyma cells around solid lignified cells can also become lignified cells by accumulating lignin and cellulose,and eventually form lignified cell clusters.In addition,it is believed that the development of a single lignified cell and the postharvest lignification of loquat fruit are two separate processes.2.The homologous transient expression system of loquat cells was constructed,the distribution and transport of lignin synthesis-related coding genes and transcription factors at the cellular level during the transient expression were studied.The homologous transient expression of Ej MYB1 and Ej4CL1 in loquat pollen and fruit was realized by using gene gun transient transformation technology combined with fluorescent protein fusion.The subcellular localization of the protein in loquat pollen showed that the fluorescence signal of Ej MYB1 protein was distributed in the nucleus,while the fluorescence signal of Ej4CL1 protein was distributed in the cytoplasm.During the germination of loquat pollen,the fluorescence signal of Ej4CL1 protein in the pollen grains decreased,and gradually moved to the top area of the actively growing pollen tube,indicating that the Ej4CL1protein was mainly concentrated in the cell area where the cell-wall lignin synthesis was vigorous.The transient expression of Ej4CL1 protein in loquat fruit cells showed that the signal mainly began to accumulate on the cell close to one side of the cell wall,indicating that the accumulation of lignin in the loquat cell wall may not be synchronized across the cell wall and may have spatial heterogeneity.3.The key pectin composition involved in the postharvest lignification of loquat fruit was screened at the cellular level,and the cell-wall dynamic mechanism behind was proposed.Based on immunofluorescence histochemistry technology,an in-situ labeling system for cell wall compositions of loquat fruit was constructed,and the content,structure and distribution of cellulose,hemicellulose,and pectin in parenchyma cells,vascular bundles and exocarp tissues during postharvest storage of loquat fruit were studied.Especially,low methylesterified homogalacturonan（HG）recognized by JIM5antibody,low methylesterified HG was found gradually accumulate in the middle lamella and cell-wall corner of loquat fruit during postharvest lignification,and trend of JIM5 signal intensity was significantly positively correlated with fruit firmness.Furthermore,it was found that the PME activity was significantly increased in the0°C group at the early storage stages,and the calcium pectate content continued to increase during the storage period,indicating that the HG was demethylesterified by PME and combined with Ca²⁺to form“egg box”structure.The formation and distribution of egg box in the loquat fruit was determined based on the OG probe labeling.Through exogenous injection to simulate the physiological conditions of fruit texture deterioration,it was found that the firmness of the loquat fruit in the PME+Ca²⁺injection group was significantly higher than that of the control group,and the JIM5 antibody and OG probe labeling indicated that the loquat fruit in the PME+Ca²⁺injection group had more low methylesterified HG and egg box structure.These results indicate that PME-mediated demethylesterification of HG occurs in the postharvest lignification of loquat fruit,and promotes the accumulation of low methylesterified HG in the middle lamella and cell-wall corner,then through further binding with Ca²⁺,egg box structure is formed,which ultimately leads to an increase in fruit firmness.It is believed that low methylesterified HG may be an important cell-wall composition that involved in postharvest lignification of loquat fruit.