| Tomato is a fleshy berry with high nutritional and economic value.It is one of the most important economic vegetables in the world.However,the tomato has severe fruit cracking after the color-breaking period,which brings huge difficulties to storage,transportation,production and sales,and no effective solution has been found yet.Fruit softening is an important factor affecting fruit cracking.As an important sign of fruit maturity,the degree of fruit softening determines the quality of the fruit.The most important change in the cell wall during the fruit softening process is the degradation of pectin material.The reduction of pectin makes the adjacent cells lose their connection and separate from each other,and finally soften the fruit.In addition,a lot of soil in southern of China is acidic and contains a large amount of metal ions,which brings serious harm to human production and life.Pectin naturally carries a variety of negatively charged functional groups,and can combine with positively charged metal ions in the surrounding environment,thereby reducing metal pollution in the soil and water environment.Therefore,increasing the pectin content is one of the effective ways to solve these problems of tomato easy cracking,intolerance to storage and transportation,and improvement of acidic soil in the southern China.But so far,many core problems in regulating the biological synthesis of pectin still need to be solved urgently.Therefore,it is very necessary to choose tomato as a research plant to study the regulation of pectin biosynthesis.UDP-glucuronic acid 4-epimerase(abbreviated as UGlcAE)can catalyze interconversion between uridine diphosphate-glucuronic acid(UDP-Glc A)and phosphate-galacturonic acid(UDP-Gal A).It is a key enzyme that regulates pectin biosynthesis.So far,the research of UGlcAE in Solanaceae plants is still blank,and the research of UGlcAE has focused on gene expression and physiological,biochemical,and enzymatic properties.In-depth functional studies of UGlcAE family genes are rarely reported.In view of this,this paper uses tomato as the research object.Based on the study of the phylogeny and expression pattern of the UGlcAE gene family,the function of the UGlcAE1 gene in tomato development is explored,and the effect of tomato UGlcAE1 in regulating pectin on fruit softening is analyzed.The physiological mechanism lays a theoretical foundation for the cultivation of tomato varieties with high pectin content in the future.The main research results obtained are as follows:1.Identification and phylogenetic analysis of tomato UGlcAE gene familyThe UGlcAE family contains 6 subfamilies.There are 9 UGlcAE family members in tomato.These 9 UGlcAE gene family members are divided into 5 subfamilies,which are distributed on 8 tomato chromosomes.Phylogenetic analysis shows that the sequence of UGlcAE orthologous genes is highly conserved.Nine tomato UGlcAE genes and six Arabidopsis UGlcAE genes are all single-exon structures.The UGlcAE gene in Arabidopsis is involved in cell components,molecular functions and biological processes,while the UGlcAE gene in tomato is only involved in molecular functions and biological processes.The amino acid sequences and promoter regions of tomato UGlcAE gene family members are very conserved during evolution.The cis-acting element upstream(0.5kb)of the tomato UGlcAE gene contains 3 hormones,5 stress,and 5 development.According to the analysis results of cis-acting elements,the expression pattern analysis of IAA,GA and SA three plant hormone-treated tomatoes showed that the prediction of the response sites of these three hormones was relatively reliable.As the tomato fruit matures,its hardness gradually decreases.The content of soluble pectin(WSP)shows an upward trend in the early stage,reaches the maximum in the MG stage,and then decreases.During tomato fruit development,the expression levels of 4 genes(UGlcAE1,UGlcAE1-like,UGlcAE5 and UGlcAE6)were relatively high,and the expression levels of the other 5 genes were low.2.Effect of UGlcAE1 gene on pectin content and related traitsThe UGlcAE1 overexpression vector was constructed,and UGlcAE1 gene overexpression tomato plants were obtained by screening.Compared with wild-type tomato,the expression level of UGlcAE1 in UGlcAE1 gene overexpression tomato was significantly increased.Overexpression of UGlcAE1 can significantly increase the pectin content in tomato fruits and tissues.Moreover,the measurement results show that pectin is mainly present in the fruit,and the content in other tissues is significantly lower than the pectin content in the fruit.The cracking rate of UGlcAE1 overexpression tomato fruits is significantly lower than that of the wild type,and with the passage of time,the improvement of the cracking rate has become more and more obvious.Overexpression of UGlcAE1 can also significantly increase the firmness of tomato fruits.The inhibitory effect of cadmium and aluminum ions on the expression level of Sl UGlcAE1 gene in the underground parts of overexpression plants was more obvious than that on the above-ground parts.In addition,the mutation of Arabidopsis UGlcAE1 gene can significantly reduce the pectin content.The petiole length and leaf length of the UGlcAE1 mutant plants were significantly smaller than those of the wild type.However,the germination rate,plant height,number of pods per plant,and pod length of the UGlcAE1 mutant did not change significantly compared with the wild type.Low-concentration cadmium ion treatment will increase the pectin content.As the cadmium ion concentration increases,the promotion effect gradually weakens and becomes an inhibitory effect.3.Transcriptome and metabolome joint analysis of the effect of UGlcAE1 gene overexpression on tomato pectin accumulationTranscriptome sequencing detected 1,620 differentially expressed genes,accounting for 4.75% of the total detected genes.Among these genes,10 genes related to pectin were screened,and 1 gene related to pectin synthesis was Solyc02g087350.3(galactosyltransferase-like 10,increased expression level);and 5 pectin degradation-related genes,namely Solyc03g112170.1(pectinase inhibitor-like,expression level increased),Solyc03g112970.1(pectinase 1-like,expression level decreased),Solyc03g123630.4(pectinase Inhibitor U1 precursor,the expression level is increased),Solyc04g076650.2(rhamnogalacturonic acid lyase B-like,the expression level is decreased)and Solyc12g096730.3(polygalacturonase,the expression level is decreased);and 1 gene related to pectin resistance to metal pollution is Solyc01g005470.3(plant anti-cadmium protein,the expression level is increased);2genes related to pectin participating in the formation of cell walls are Solyc07g065660.4(cellulose synthase-like protein E1,the expression level is elevated),and Solyc09g072820.4(cellulose synthase A catalytic subunit 4,the expression level is elevated);1 gene related to the involvement of pectin in fruit ripening is Solyc04g072160.3(maturation regulatory protein)DDTFR8,the expression level is elevated).The overexpression of UGlcAE1 may lead to the enhancement of the catalytic effect of galactosyltransferase in the process of pectin synthesis,and the decrease of the activities of rhamnogalacturonic acid lyase,pectinase and polygalacturonase and other pectin-degrading enzymes,and the increase of the activity of the precursors of pectinase inhibitors and pectinase inhibitors,which eventually caused the increase of pectin content.The results of metabolome sequencing showed that there were 1094 metabolites measured in the positive ion mode and 708 metabolites measured in the negative ion mode,all of which are known metabolites.Through the analysis of differential metabolites,a total of 3 metabolites related to pectin synthesis(uridine diphosphate galactose,xylulose and glucose 6-phosphate)were screened.They were reduced by7.82%,11.76%,and 93.622% respectively in UGlcAE1 overexpression tomatoes.Combined with the transcriptome analysis results,the overexpression of UGlcAE1 can cause the reduction of three substances(uridine diphosphate galactose,xylulose and glucose 6-phosphate)and one encoding enzyme(galactosyltransferase)during the process of pectin synthesis.The increase in gene expression levels of enzymes is presumably due to their joint action leading to an increase in pectin synthesis,which leads to an increase in pectin content. |
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