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Starch degradation plays a central role in fruit ripening. The β-amylase (Bam) catalysts starch degradation of apple fruit. However, the molecular mechanism regulating Bam gene expression in apples remains unclear. Using yeast one-hybrid library screening, we identified a transcription factor MdGTL1a that directly binds to the MdBam8 promoter. This bind was verified by using an electrophoretic mobility shift assay and confirmed to enhance the promotor activity of MdBam8 by promoter-β-glucuronidase transactivation assay. Transient over-expression of MdGTL1a activated MdBam8 expression and further enhanced starch degradation in apple flesh. Subcellular localization analyses in tobacco protoplasts demonstrated that the nucleus was the exclusive location of the MdGTL1a-GFP fusion protein. Exogenous salicylic acid treatment decreased MdGTL1a expression and resulted in higher starch content in apples, which was consistent with the fruit ripening. It is concluded that salicylic acid treatment could enhance apple storage quality by inhibiting starch degradation through a crucial transcription factor, MdGTL1a., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier B.V. All rights reserved.)

Apple replant disease (ARD) is a worldwide problem that threatens the industry. However, the genetic mechanism underlying plant disease resistance against ARD remains unclear. In this study, a negative regulatory microRNA in Malus domestica, mdm-miR397b, and its direct target MdLAC7b (Laccase) was selected for examination based on our previous small RNA and degradome sequencing results. Overexpressing the mdm-miR397b-MdLAC7b module altered the lignin deposition and jasmonic acid contents in apple roots, which also led to increased resistance to Fusarium solani. Additionally, Y1H library screening using mdm-miR397b promoter recombinants identified a transcription factor, MdERF61, that represses mdm-miR397b transcriptional activity by directly binding to 2 GCC-boxes in the mdm-miR397b promoter. In summary, our results suggest that the MdERF61-mdm-miR397b-MdLAC7b module plays a crucial role in apple resistance to F. solani and offers insights for enhancing plant resistance to soil-borne diseases in apples., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Flavonols are widely synthesized throughout the plant kingdom, playing essential roles in plant physiology and providing unique health benefits for humans. Their glycosylation plays significant role in improving their stability and solubility, thus their accumulation and function. However, the genes encoding the enzymes catalyze this glycosylation remain largely unknown in apple. This study utilized a combination of methods to identify genes encoding such enzymes. Initially, candidate genes were selected based on their potential to encode UDP-dependent glycosyltransferases (UGTs) and their expression patterns in response to light induction. Subsequently, through testing the in vitro enzyme activity of the proteins produced in Escherichia coli cells, four candidates were confirmed to encode a flavonol 3-O-galactosyltransferase (UGT78T6), flavonol 3-O-glucosyltransferase (UGT78S1), flavonol 3-O-xylosyltransferase/arabinosyltransferase (UGT78T5), and flavonol 3-O-rhamnosyltransferase (UGT76AE22), respectively. Further validation of these genes' functions was conducted by modulating their expression levels in stably transformed apple plants. As anticipated, a positive correlation was observed between the expression levels of these genes and the content of specific flavonol glycosides corresponding to each gene. Moreover, overexpression of a flavonol synthase gene, MdFLS, resulted in increased flavonol glycoside content in apple roots and leaves. These findings provide valuable insights for breeding programs aimed at enriching apple flesh with flavonols and for identifying flavonol 3-O-glycosyltransferases of other plant species., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

To maximize breeding and exploitation of disease resistance traits for managing apple replant disease (ARD), it is of great importance to understand the mechanisms of apple root resistance. Currently, little is known about the functions of the specific genes that confer resistance traits in apple root. In this study, molecular, biochemical, and genetic approaches allowed an in-depth understanding of the role of the MdPR4 gene in the defense response of apple root. The MdPR4 encoding gene showed upregulation following ARD pathogen inoculation in our previous transcriptome data. Subcellular localization analyses revealed that MdPR4 is localized on the plasma membrane, endoplasmic reticulum, and apoplast, which is mainly determined by its signal peptide. Molecular docking analysis between MdPR4 protein with chitin molecule and in vitro MdPR4 chitin affinity assay proved its chitin-binding ability, which provided evidence for its role in chitin-mediated immune responses. Purified MdPR4 protein and MdPR4 overexpressed apple callus inhibited spore germination and mycelial growth of ARD-related Fusarium spp. pathogens. These data support the conclusion that MdPR4 is a chitin-binding protein in apple vegetative tissues that may play an important role in defense activation in response to ARD pathogen infection., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacts with VemR. Phos-tag SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection., (© 2020 College of Life Science and Technology, Guangxi University. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

The aroma quality of citrus fruit is determined by volatiles that are present at extremely low levels in the citrus fruit juice sacs; it can be greatly improved by increasing volatiles. In this study, we showed that the contents of cis - and trans -linalool oxides were significantly increased in the juice sacs of three pummelos artificially pollinated with the Citrus mangshanensis (MS) pollen. A novel cytochrome P450 78A7 gene ( CitLO 1) was significantly upregulated in the juice sacs of Huanong Red pummelo pollinated with MS pollen in comparison to that with open pollination. Compared to wild-type tobacco Bright-Yellow2 cells, transgenic cells overexpressing CitLO 1 promoted a 3- to 4-fold more conversion of (-)-linalool to cis - and trans -linalool oxides. Overall, our results suggest that MS pollen has a xenia effect on pummelo fruit aroma quality, and CitLO 1 is a linalool oxide synthase gene that played an important role in the xenia effect.

Ethylene response factors (ERFs) are known to regulate fruit ripening. However, the ERF regulatory networks are not clear. In this study, we have shown that peach (Prunus persica) PpeERF2 regulates fruit ripening through suppressing the expression of two ABA biosynthesis genes (PpeNCED2, PpeNCED3) and a cell wall degradation gene (PpePG1). The transcript levels of PpeERF2 in fruit were opposite to that of PpeNCED2, PpeNCED3 and PpePG1 during ripening and in response to various ripening treatments. PpeERF2 was found to bind to the PpeNCED2, PpeNCED3 and PpePG1 promotors as demonstrated by yeast one-hybrid (Y1H) and EMSA assays; and further found to repress the promoter activities of the three genes in tobacco leaf tissues after Agrobacterium infiltration. Taken together, these results provide new information for a better understanding of the crosstalk network between ethylene signaling, cell wall degradation and ABA biosynthesis during fruit ripening., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Grapevine is an important fruit crop grown worldwide, and its cultivars are mostly derived from the European species Vitis vinifera , which has genes for high fruit quality and adaptation to a wide variety of climatic conditions. Disease resistance varies substantially across grapevine species; however, the molecular mechanisms underlying such variation remain uncharacterized., Results: The anatomical structure and disease symptoms of grapevine leaves were analyzed for two grapevine species, and the critical period of resistance of grapevine to pathogenic bacteria was determined to be 12 h post inoculation (hpi). Differentially expressed genes (DEGs) were identified from transcriptome analysis of leaf samples obtained at 12 and 36 hpi, and the transcripts in four pathways (cell wall genes, LRR receptor-like genes, WRKY genes, and pathogenesis-related (PR) genes) were classified into four co-expression groups by using weighted correlation network analysis (WGCNA). The gene VdWRKY53 , showing the highest transcript level, was introduced into Arabidopsis plants by using a vector containing the CaMV35S promoter. These procedures allowed identifying the key genes contributing to differences in disease resistance between a strongly resistant accession of a wild grapevine species Vitis davidii ( VID ) and a susceptible cultivar of V. vinifera , 'Manicure Finger' ( VIV ). Vitis davidii, but not VIV, showed a typical hypersensitive response after infection with a fungal pathogen ( Coniella diplodiella ) causing white rot disease. Further, 20 defense-related genes were identified, and their differential expression between the two grapevine species was confirmed using quantitative real-time PCR analysis. VdWRKY53 , showing the highest transcript level, was selected for functional analysis and therefore over-expressed in Arabidopsis under the control of the CaMV35S promoter. The transgenic plants showed enhanced resistance to C. diplodiella and to two other pathogens, Pseudomonas syringae pv. tomato DC3000 and Golovinomyces cichoracearum ., Conclusion: The consistency of the results in VID and transgenic Arabidopsis indicated that VdWRKY53 might be involved in the activation of defense-related genes that enhance the resistance of these plants to pathogens. Thus, the over-expression of VdWRKY53 in transgenic grapevines might improve their resistance to pathogens., Competing Interests: Not applicable.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Stone cells negatively affect fruit quality because of their firm and lignified cell walls, so are targets for reduction in pear breeding programmes. However, there is only limited knowledge of the molecular mechanisms underlying the formation of stone cells. Here, we show that PbrMYB169, an R2R3 MYB transcription factor, of Pyrus bretschneideri positively regulates lignification of stone cells in pear fruit. PbrMYB169 was shown to be co-expressed with lignin biosynthesis genes during pear fruit development, and this co-expression pattern was coincident with stone cell formation in the fruit of Pyrus bretschneideri 'Dangshansuli'. The PbrMYB169 expression level was also positively correlated with stone cell content in 36 pear cultivars tested. PbrMYB169 protein significantly activated the promoter of lignin genes C3H1, CCR1, CCOMT2, CAD, 4CL1, 4CL2, HCT2, and LAC18 via binding to AC elements [ACC(T/A)ACC] in these promoters. Furthermore, overexpression of PbrMYB169 in transgenic Arabidopsis plants enhanced the expression of lignin genes, and increased lignin deposition and cell wall thickness of vessel elements, but did not change the ratio of syringyl and guaiacyl lignin monomers. In conclusion, PbrMYB169 appears to be a transcriptional activator of lignin biosynthesis and regulates secondary wall formation in fruit stone cells. This study advances the understanding of the regulation of lignin biosynthesis and provides valuable molecular genetic information for reducing stone cell content in pear fruit., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Flowering plants utilize different floral structures to develop flesh tissue in fruits. Here we show that suppression of the homeologous SEPALLATA1/2-like genes MADS8 and MADS9 in the fleshy fruit apple (Malus x domestica) leads to sepaloid petals and greatly reduced fruit flesh. Immunolabelling of cell-wall epitopes and differential staining showed that the developing hypanthium (from which the apple flesh develops) of MADS8/9-suppressed apple flowers lacks a tissue layer, and the remaining flesh tissue of fully developed apples has considerably smaller cells. From these observations, it is proposed that MADS8 and MADS9 control the development of discrete zones within the hypanthium tissue, and therefore fruit flesh, and also act as foundations for development of different floral organs. At fruit maturity, the MADS8/9-suppressed apples do not ripen in terms of both developmentally controlled ripening characters, such as starch degradation, and ethylene-modulated ripening traits. Transient assays suggest that, like the RIN gene in tomato, the MADS9 gene acts as a transcriptional activator of the ethylene biosynthesis enzyme, 1-aminocyclopropane-1-carboxylate (ACC) synthase 1. The existence of a single class of genes that regulate both flesh formation and ripening provides an evolutionary tool for controlling two critical aspects of fleshy fruit development., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

In many plant species, exposure to a prolonged period of low temperature during the winter promotes flowering in the spring, a process termed vernalization. In Arabidopsis, the vernalization requirement of winter annual ecotypes is caused by a MADS-box gene FLOWERING LOCUS C (FLC), which is a repressor of flowering gene. Here, a MADS-box gene was isolated from an early flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata L. Raf) by the RACE method combined with a cDNA library. Phylogenetic analysis reveals that the MADS-box gene is more closely related to the homologs of the FLOWERING LOCUS C lineage than to any of the other MIKC-type MADS-box lineages known from Arabidopsis. The expression profile of the MADS-box gene by real-time PCR showed upregulation of PtFLC during the winter, followed by a decrease in the spring and summer. This kind of cycling is contrary to the pattern observed in Arabidopsis. In situ hybridization reveals that the MADS-box gene is predominately expressed in the vegetative and reproductive meristems. In addition, five alternatively spliced transcripts of the MADS-box gene were also isolated at juvenile and adult mutant developmental stages. Expression analysis of these transcripts at different developmental stages indicated involvement of alternative splicing during phase change. The information suggests a complicated regulation mechanism in seasonal response and flower formation in perennial woody plants.

To increase our understanding of the genes involved in flower development in citrus, we identified genes differentially expressed between juveniles and adults of an early-flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.) by suppressive subtractive hybridization (SSH) and macroarray hybridization analyses. Based on dot blot analysis, we confirmed that the juvenile subtracted cDNA library comprised genes upregulated during floral induction, inflorescence development and flowering. A total of 190 nonredundant expressed sequence tags were identified that were related flower development. The temporal and spatial expression patterns of four SSH-enriched genes were studied in adult precocious trifoliate orange by real-time PCR. Among these differentially expressed genes, the expression pattern of C5-B16 was closely correlated with inflorescence development and flowering. The full-length cDNA of C5-B16 was isolated by 5' and 3' rapid amplification of cDNA ends. Sequence analysis indicated that C5-B16 is a novel gene in citrus.

The phosphorylation-specific peptidyl prolyl cis/trans isomerase (PPIase) Pin1 in humans and its homologues in yeast and animal species play an important role in cell cycle regulation. These PPIases consist of an NH(2)-terminal WW domain that binds to specific phosphoserine- or phosphothreonine-proline motifs present in a subset of phosphoproteins and a COOH-terminal PPIase domain that specifically isomerizes the phosphorylated serine/threonine-proline peptide bonds. Here, we describe the isolation of MdPin1, a Pin1 homologue from the plant species apple (Malus domestica) and show that it has the same phosphorylation-specific substrate specificity and can be inhibited by juglone in vitro, as is the case for Pin1. A search in the plant expressed sequence tag data bases reveals that the Pin1-type PPIases are present in various plants, and there are multiple genes in one organism, such as soybean (Glycine max) and tomato (Lycopersicon esculentum). Furthermore, all these plant Pin1-type PPIases, including AtPin1 in Arabidopsis thaliana, do not have a WW domain, but all contain a four-amino acid insertion next to the phospho-specific recognition site of the active site. Interestingly, like Pin1, both MdPin1 and AtPin1 are able to rescue the lethal mitotic phenotype of a temperature-sensitive mutation in the Pin1 homologue ESS1/PTF1 gene in Saccharomyces cerevisiae. However, deleting the extra four amino acid residues abolished the ability of AtPin1 to rescue the yeast mutation under non-overexpression conditions, indicating that these extra amino acids may be important for mediating the substrate interaction of plant enzymes. Finally, expression of MdPin1 is tightly associated with cell division both during apple fruit development in vivo and during cell cultures in vitro. These results have demonstrated that phosphorylation-specific PPIases are highly conserved functionally in yeast, animal, and plant species. Furthermore, the experiments suggest that although plant Pin1-type enzymes do not have a WW domain, they may fulfill the same functions as Pin1 and its homologues do in other organisms.

Differential display was used to isolate genes differentially expressed early in fruit development of apple (Malus domestica Borkh.). This approach resulted in the isolation of MDH1, a homeobox gene with a homeodomain similar to that of BELL1 (BEL1), which is involved in regulation of ovule development in Arabidopsis. However, outside the homeodomain MDH1 is quite different from BEL1. In apple, MDH1 mRNA was predominantly found in flowers, expanding leaves and expanding fruit. In pre-anthesis flowers, in situ hybridization showed that MDH1 mRNA accumulated in ovules. To further investigate the function of this new homeobox gene, MDH1 was transformed into Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. The transgenic Arabidopsis plants showed dwarfing, reduced fertility and changes in carpel and fruit (silique) shape. The size and shape of the cells in the transgenic fruit was irregular. Both the transgenic phenotypes in Arabidopsis and the expression pattern of this gene in apple are consistent with the idea that MDH1 is likely to play an important role in control of plant fertility.