PbrmiR397a regulates lignification during stone cell development in pear fruit

Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA element that responded to salicylic acid to induce gene expression as confirmed using a cell‐based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl‐ and guaiacyl‐lignin monomers. Consistent with reduction in lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild‐type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality.     

Characterization of a cinnamoyl-CoA reductase that is associated with stem development in wheat

Cinnamoyl-CoA reductase (CCR) is responsible for the CoA ester to aldehyde conversion in monolignol biosynthesis, which diverts phenylpropanoid-derived metabolites into the biosynthesis of lignin. To gain a better understanding of lignin biosynthesis and its biological function, a cDNA encoding CCR was identified from wheat (Triticum aestivum L.), and designated as Ta-CCR1. Phylogenetic analysis indicated that Ta-CCR1 grouped together with other monocot CCR sequences while it diverged from Ta-CCR2. DNA gel-blot and mapping analyses demonstrated that Ta-CCR1 is present as a single copy gene in the wheat genome. Recombinant Ta-CCR1 protein converted feruloyl CoA, 5-OH-feruloyl CoA, sinapoyl CoA, and caffeoyl CoA, but feruloyl-CoA was the best substrate, suggesting the preferential biosynthesis of G-type lignin. RNA gel-blot analysis indicated that Ta-CCR1 was highly expressed in stem, with lower expression in leaves, and undetectable expression in roots. CCR enzyme activity was increased progressively along with the lignin biosynthesis and stem maturity. During stem development, Ta-CCR1 mRNA levels remained high at elongation, heading, and milky stages in the wheat H4564 cultivar, while they declined dramatically at the heading and milky stages in stems of the C6001 cultivar. Ta-CCR1 mRNA expression paralleled extractable CCR enzyme activity in these two cultivars. Furthermore, high Ta-CCR1 mRNA levels and high CCR enzyme activity in wheat stem were correlated with a higher Klason lignin content and greater stem mechanical strength in the H4564 cultivar. This suggests that Ta-CCR1 and its related CCR enzyme may be involved in the regulation of lignin biosynthesis during stem maturity and then contributes to stem strength support in wheat.     

Functional analysis of four Class III peroxidases from Chinese pear fruit: a critical role in lignin polymerization

Pear fruit could be used as good medicine to relieve coughs, promote salivation, nourish lungs, and reduce the risk of many diseases for its phytochemical action. Lignin is a major secondary metabolite in Chinese pear fruit. Class III peroxidase (Class III PRX) is an important enzyme in the biosynthesis of lignin in plants. However, we poorly understand the role of PRXs in lignin biosynthesis in Chinese pear fruit. In our study, we cloned five PRXs from Chinese pear (Pyrus bretschneideri), namely PbPRX2, PbPRX22, PbPRX34, PbPRX64, and PbPRX75, which contained 978 bp encoded 326 amino acids (AA), 2607 bp encoded 869 AA, 972 bp encoded 324 AA, 687 bp encoded 229 AA, and 1020 bp encoded 340 AA, respectively. Enzyme activity analysis showed that four recombinant PbPRX proteins had catalytic activities for pyrogallol, guaiacol, ferulic acid, coniferyl alcohol, and sinapyl alcohol. Subcellular localization experiments showed that these genes were located in the cell wall or cell membrane. Enzyme activity and kinetics of PbPRX2 revealed its role in polymerization of lignin in Chinese pear fruit. The present study suggested that PbPRXs played critical roles in lignin biosynthesis in Chinese pear fruit.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00949-9.     

Roles of gibberellins in increasing sink demand in Japanese pear fruit during rapid fruit growth

Our previous work demonstrated that exogenous gibberellins (GAs) applications during rapid fruit growth significantly increases sink demand and results in a larger fruit in Japanese pear. In an attempt to unravel the mechanism of increased sink demand by applied GAs, the histology, cell wall components of the flesh, and carbon accumulation in the fruit were assessed for Japanese pear (Pyrus pyrifolia, cultivar ‘Kousui’), as were the activities of sucrose- and sorbitol-cleaving enzymes. Our results show that most vascular tissues occurred in core tissue with very little vascular tissue in the flesh. Application of a mixture of GA₃ + GA₄ in lanolin paste significantly increased the amount of ethanol-insoluble solids, e.g., total pectins, hemicellulose, and cellulose in the cell walls. There was a significantly increased sink demand (assessed by ¹³C accumulation in the fruit) by the applied GAs, and this increased sink strength was closely related to increased activities of cell wall-bound invertase in the core, neutral invertase and NAD-dependent sorbitol dehydrogenase in the flesh during rapid fruit growth. As well, concentrations of sorbitol and sucrose in the flesh were decreased by GA application, while glucose concentration increased. Most importantly, the fact that sink activity can be increased by GA application implies that endogenous GAs are likely to be important modulators for sugar metabolism. Hence, selecting for genotypes with elevated GA production in the growing fruit and increased activities of key enzymes for sugar metabolism could result in increased fruit size.     

Molecular cloning and expression of squalene synthase and 2,3-oxidosqualene cyclase genes in persimmon (Diospyros kaki L.) fruits

Oleanolic acid (OA) and ursolic acid (UA) are the main triterpene acids in persimmon fruit, and squalene synthase and 2,3-oxidosqualene cyclases are important enzymes in pentacyclic triterpene biosynthesis. In order to study their relationship, DkSQS and DkOSC were cloned from persimmon fruits in the present study. The full-length cDNA of DkSQS was 1647 bp, containing an open reading frame (ORF) of 1245 bp that encoded a peptide of 415 amino acids (AA). The 3′-end of DkOSC cDNA fragment contained 522 bp, including a partial ORF of 298 bp, a full poly A tail that encoded 98 AA. Two cultivars of persimmon, i.e. cv. Nishimurawase and cv. Niuxinshi, were used to study the content of OA and UA and the related gene expression. Results showed that OA and UA contents changed in both cultivars during fruit development, the difference in cv. Nishimurawase was greater than that in cv. Niuxinshi. The expression of DkSQS and DkOSC had no obvious correlation with the biosynthesis of OA and UA in the flesh. There may be two main reasons. Firstly, different enzymes involved in the biosynthesis of triterpenes and mutual adjustment were existed in different gene expressions. Secondly, it was not clear that the DkOSC cloned in this research belonged to which subfamily. Therefore, the real relationship between triterpenes and DkSQS and DkOSC in persimmon fruits is still to be revealed.     

Redirection of the phenylpropanoid pathway to feruloyl malate in <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants deficient for cinnamoyl-CoA reductase 1

Cinnamoyl-CoA reductase 1 (CCR1, gene At1g15950) is the main CCR isoform implied in the constitutive lignification of Arabidopsis thaliana. In this work, we have identified and characterized two new knockout mutants for CCR1. Both have a dwarf phenotype and a delayed senescence. At complete maturity, their inflorescence stems display a 25–35% decreased lignin level, some alterations in lignin structure with a higher frequency of resistant interunit bonds and a higher content in cell wall-bound ferulic esters. Ferulic acid-coniferyl alcohol ether dimers were found for the first time in dicot cell walls and in similar levels in wild-type and mutant plants. The expression of CCR2, a CCR gene usually involved in plant defense, was increased in the mutants and could account for the biosynthesis of lignins in the CCR1-knockout plants. Mutant plantlets have three to four-times less sinapoyl malate (SM) than controls and accumulate some feruloyl malate. The same compositional changes occurred in the rosette leaves of greenhouse-grown plants. By contrast and relative to the control, their stems accumulated unusually high levels of both SM and feruloyl malate as well as more kaempferol glycosides. These findings suggest that, in their hypolignified stems, the mutant plants would avoid the feruloyl-CoA accumulation by its redirection to cell wall-bound ferulate esters, to feruloyl malate and to SM. The formation of feruloyl malate to an extent far exceeding the levels reported so far indicates that ferulic acid is a potential substrate for the enzymes involved in SM biosynthesis and emphasizes the remarkable plasticity of Arabidopsis phenylpropanoid metabolism.     

鸭梨PAL基因的反义遗传转化及表达分析

苯丙氨酸解氨酶(phenylalanin ammonia-lyase,PAL,EC4.3.1.5)是植物通过苯丙烷代谢途径合成木质素的关键酶和限速酶,其通过影响木质素的合成而与果实中石细胞的分化、发育及果实品质密切相关。为了降低鸭梨中苯丙氨酸解氨酶的含量,该研究利用反义PAL基因遗传转化鸭梨、降低鸭梨内源PAL基因的表达。结果表明:(1)采用RT-PCR技术,利用根据Gen Bank中西洋梨PAL基因序列设计特异性引物,扩增得到496 bp的鸭梨PAL基因片段。(2)将扩增片段反向插入载体p BI121的MCS区域,构建植物PAL基因反义表达载体p BI121-As PAL。接着采用电转化法将反义表达载体转入农杆菌EHA105中,并制备出农杆菌工程菌液。(3)利用农杆菌介导法对鸭梨组培苗叶片外植体进行遗传转化,得到23株转基因鸭梨苗。PCR检测证实PAL反义基因片段转入鸭梨中,实时定量PCR检测表明转基因鸭梨苗体内PAL基因表达量均有所降低,为非转基因苗的65%~75%。该研究结果表明利用反义RNA技术获得了抑制内源性PAL基因表达的转基因鸭梨植株,为改善鸭梨果实品质、改良品种奠定了基础。     

Production of Host‐selective SV‐toxins by Stemphylium sp. Causing Brown Spot of European Pear in Japan

Recently, a new disease known as ‘brown spot of European pear’ caused by Stemphylium sp. appeared on the leaves, twigs and fruits of the cultivar Le Lectier in Niigata Prefecture, Japan. Because Stemphylium vesicarium (teleomorph: Pleospora allii), which causes a similar disease in Europe, has been shown to produce host‐selective SV‐toxins in culture filtrates (CFs), SV‐toxin production by Stemphylium sp. in Japan was investigated. In pathogenicity tests, the pathogen induced severe necrotic spots on the leaves of the European pear cultivar Le Lectier, slight spots on cultivar La France and slight or no spots on cultivar Bartlett. The Japanese pear cultivar Nijisseiki was not affected by the pathogen. Culture filtrates of the pathogen were tested for phytotoxicity on cultivars by a leaf necrosis assay. The sensitivity of cultivars to the CFs was consistent with the susceptibility of cultivars to the pathogen infection, indicating the presence of host‐selective toxins. The toxins were purified from the CFs according to the procedure reported for SV‐toxin purification in S. vesicarium. The results indicated that Stemphylium sp. in Japan produces the same SV‐toxins as S. vesicarium in Europe.