Apple russeting as seen through the RNA-seq lens: strong alterations in the exocarp cell wall
ABSTRACT: Russeting is a commercially important defect in apple (Malus x domestica) fruit production. Apple russeting is mainly characterized by the accumulation of suberin on the inner part of the cell wall. However, knowledge on the underlying genetic components triggering this trait remains sketchy. A bulk transcriptomic profiling was performed on the exocarps of three russeted and three waxy apple varieties using RNA sequencing. This experimental design was chosen to lower the specificities of each genotype. A qPCR validation was carried out on representative genes and additional contrasting varieties. Gene ontology enrichment revealed a repression of the lignin and cuticle biosynthesis genes in the russeted exocarps, concomitantly with an enhanced expression of suberin deposition, stress responsive, primary sensing, NAC and MYB-family transcription factors, and specific triterpene biosynthetic genes. Notably, a strong correlation (R2=0.976) between the expression of a MYB93-like transcription factor and key suberin biosynthetic genes was found. Our results suggest that russeting is induced by a decreased expression of the cuticle layer biosynthetic genes, leading to a stress response which not only affects suberin deposition, but also the entire structure of the cell wall. In addition, the large number of candidate genes highlighted in this study provides a solid platform for further functional investigations. In order to draw a consistent picture of the gene expression profiles specific to both russeted and waxy apples and at the same time to highlight and interpret the mechanism leading to the russeted phenotype, a bulk RNA-sequencing was performed on the exocarp of a group of three distinct fully-russeted apple varieties ('Patte de loup', 'Reinette Parmentier', 'St Edmund's Pippin') and a second one including 3 fully waxy varieties ('Gala', 'CRAW/Ma/AF42', and 'CRAW/Ma/AG94').
Project description:• A comparison of the transcriptomes of russeted vs. waxy apple exocarps previously highlighted a tight relationship between a gene encoding a MYB-type transcription factor, MdMYB93, and some key suberin biosynthetic genes. The present work assesses the role of this transcription factor in the suberization process. • A phylogenetic analysis of MdMYB93 and Arabidopsis thaliana MYBs was performed and the function of MdMYB93 was further investigated using Agrobacterium-mediated transient overexpression in Nicotiana benthamiana leaves. An RNA-Seq analysis was performed to highlight the MdMYB93-regulated genes. UPLC-TripleTOF and GC-MS were used to investigate alterations in phenylpropanoid, soluble free lipid, and lipid polyester contents. • A massive accumulation of suberin and its biosynthetic precursors in MdMYB93 agro-infiltrated leaves was accompanied by a remobilization of phenylpropanoids and an increased amount of lignin precursors. Gene expression profiling displayed a concomitant alteration of lipid and phenylpropanoid metabolism, cell wall development, and extracellular transport, with a large number of induced transcripts predicted to be involved in suberin deposition. • The present work supports a major role of MdMYB93 in the regulation of suberin deposition in russeted apple skins, from the synthesis of monomeric precursors, their transport, polymerization, and final deposition as suberin in primary cell wall. In order to draw a consistent picture of a gene expression profile of the MYB93 induced was performed comparing of 4 biological replicates of Nicotiana benthamiana leaves infiltrated with Pearleygate103 35S::MdMYB93 and 4 biological replicates of control plants infiltrated with P19 only.
Project description:To understand the molecular basis of viral diseases, transcriptome profiling has been widely used to correlate host gene expression change patterns with disease symptoms during viral infection in many plant hosts. We used infection of apple by Apple stem grooving virus (ASGV), which produces no disease symptoms, to assess the significance of host gene expression changes in disease development. We specifically asked the question whether such asymptomatic infection is attributed to limited changes in host gene expression. Using RNA-seq, we identified a total of 184 up-regulated and 136 down-regulated genes in apple shoot cultures permanently infected by ASGV in comparison with virus-free shoots cultures. As in most plant hosts showing disease symptoms during viral infection, these differentially expressed genes encode known or putative proteins involved in cell cycle, cell wall biogenesis, response to biotic and abiotic stress, development and fruit ripening, phytohormone function, metabolism, signal transduction, transcription regulation, translation, transport, and photosynthesis. Our data suggest that current approaches to correlate host gene expression changes under viral infection conditions to specific infection processes or disease symptom development, based on the interpretation of individual gene functions, have severe limitations. Integrative approaches that can take into account plant development stages, gene threshold levels as well as compensatory, synergistic and antagonistic effects may be necessary to develop a sound systems understanding of the biological significance of host gene expression changes during infection. Compare the transcript profiling of ASGV-infected asymptomatic apple planlets (AP-Vinfect) and virus-free apple plantlets (AP-Vfree) by deep sequencing using Illumina RNA-Seq to check whether lots of genes were modulated by ASGV infection.
Project description:Seed germination triggers a transition of growth and metabolic activities from quiescent to active statuses. Germinating seeds is a good system to study many biological and biochemical processes including hormone metabolic activities and cell wall biosynthesis. Next generation sequence technology is used to study these processes. We have examined gene transcription activities and alternative splicing events in germinating embryos We dissected barley embryos from four barley varieties at 2 time points 24 h and 48 h
Project description:Analysis of transcriptional response of virus-infected cassava and identification of putative sources of resistance for cassava brown streak disease transcriptome analysis of two varieties of cassava that differ in their level of resistance to cassava brown streak virus.
Project description:MicroRNAs are a class of endogeneously expressed non-coding small, ~21nt RNAs involved in the negative regulation of gene expression. In plants, miRNAs are known to play a critical role in developmental and metabolic pathways, as they predominantly target transcription factors. Studies in Arabidopsis and apple have shown that few microRNAs and small interfering (si) RNAs target MYB transcription factors, which are key regulators of phenylpropanoid pathway. However, it is not well-understood how miRNAs mediate regulation of MYBs to produce secondary metabolites such as anthocyanins and flavonoids. Here we show that, a cluster of abundant miRNAs target MYB transcription factors in anthocyanin rich fruits such as grapes. Using deep small RNA-sequencing we establish that grape varieties with high anthocyanin content express abundant MYB-targeting miRNAs resulting in differential expression of MYB proteins among grape varieties, thereby regulating the phenylpropanoid pathway. Overall design: Deep sequencing of small RNA of three cultivars of grapes with varying levels of anthocyanin.
Project description:MicroRNAs are a class of endogeneously expressed non-coding small, ~21nt RNAs involved in the negative regulation of gene expression. In plants, miRNAs are known to play a critical role in developmental and metabolic pathways, as they predominantly target transcription factors. Studies in Arabidopsis and apple have shown that few microRNAs and small interfering (si) RNAs target MYB transcription factors, which are key regulators of phenylpropanoid pathway. However, it is not well-understood how miRNAs mediate regulation of MYBs to produce secondary metabolites such as anthocyanins and flavonoids. Here we show that, a cluster of abundant miRNAs target MYB transcription factors in anthocyanin rich fruits such as grapes. Using RNA-sequencing we establish that grape varieties with high anthocyanin content express abundant MYB-targeting miRNAs resulting in differential expression of MYB proteins among grape varieties, thereby regulating the phenylpropanoid pathway. Overall design: RNA-Seq of three cultivars of grapes with varying levels of anthocyanin.
Project description:Closed terminal buds of apple trees (Malus x domestica Borkh, Royal Gala and Castel Gala varieties) grown in commercial orchards were harvested during autumn and winter and exposed to cold treatments 18 biological samples, consisting of 9 pairs of replicates, were analysed in dye-swap. Samples are whole closed terminal buds. Biological replicates are buds from 2 different harvest year subjected to similar cold treatments. Samples with contrasting dormancy status in the same harvest year were compared in 8 dye-swap. Most samples were hybridized more than once in different combinations
Project description:The purpose of this project is to examine the effects of rootstocks on the gene expression patterns in scions of apple trees. Gene expression patterns were examined in the Gala variety grafted onto seven different, commonly used rootstocks. These trees were grown in the greenhouse to limit environmental effects. Also, gene expression profiles were examined in three different varieties (Ambrosia, Melrose,and Gala) grafted onto B.9 rootstocks grown in the field. Each sample is a pool of RNA from two different trees. RNA samples were isolated from 0.5 g of actively growing shoot tips, including leaf and stem tissues.
Project description:The study aims essentially at the characterisation of suberin degradation mechanisms by Aspergillus nidulans, at a fundamental level. Suberin is an important protective barrier in plant, thus the study of its biodegradation significantly impacts on phytopatology. In addition, fungal suberin degrading enzymes might provide important insights to develop new waste management, bioremediation and biodeterioration prevention strategies. Overall design: The Aspergillus nidulans cultures were grown for two days in mineral media containing glucose (T0). After this time point, the extracellular media was replaced by fresh mineral media containing suberin as the sole carbon source and allowed to incubate for two (T1), four (T2) and six (T3) days. To investigate the mechanisms by which the fungus was able to degrade suberin, the fungal transcriptome (three biological replicates) was analysed using Affymetrix microarrays. Pair-wise comparisons were used to identify the set of genes with altered expression levels between the control (grown on glucose) and during growth on suberin for two, four or six days.
Project description:Background: Geographic variation in the thermal environment impacts a broad range of biochemical and physiological processes and can be a major selective force leading to local population adaptation. In the intertidal copepod Tigriopus californicus, populations along the coast of California show differences in thermal tolerance that are consistent with adaptation, i.e., southern populations withstand thermal stresses that are lethal to northern populations. To understand the genetic basis of these physiological differences, we use an RNA-seq approach to compare genome-wide patterns of gene expression in two populations known to differ in thermal tolerance. Results: Observed differences in gene expression between the southern (San Diego) and the northern (Santa Cruz) populations included both the number of affected loci as well as the identity of these loci. However, the most pronounced differences concerned the amplitude of up-regulation of genes producing heat shock proteins (Hsps) and genes involved in ubiquitination and proteolysis. Cuticle genes were up-regulated in SD but down-regulated in SC, and mitochondrial genes were downregulated in both populations. Among the hsp genes, orthologous pairs show markedly different thermal responses as the amplitude of hsp response was greatly elevated in the San Diego population, most notably in members of the hsp70 gene family. There was no evidence of accelerated evolution at the sequence level for hsp genes. Conclusions: Marked changes in gene expression were observed in response to acute sublethal thermal stress in the copepod T. californicus. Although some qualitative differences were observed between populations (e.g., cuticle gene regulation), the most pronounced differences involved the magnitude of induction of numerous hsp and ubiquitin genes. These differences in gene expression suggest that evolutionary divergence in the regulatory pathway(s) involved in acute temperature stress may offer at least a partial explanation of latitudinal trends in thermal tolerance observed in Tigriopus. For each population, ~600 copepods were split into two equal samples, one for control and one for treatment. Each sample was placed in a 50 mL Falcon tube containing 30 mL filtered seawater. After equilibrating samples to 20 degrees C, each tube was immersed in water bath at its target temperature (control: 20 C; treatment: 35 C) for one hour, and then immersed at 20 C for one hour for recovery. Copepods were then collected in a net mesh and quickly transferred to a tube containing 5 mL Tri-reagent for standard RNA extraction.