RNA-seq, de novo transcriptome assembly and flavonoid gene analysis in 13 wild and cultivated berry fruit species with high content of phenolics

This research was funded by the European Union Framework Program 7, Project BacHBerry [FP7–613793]. The authors also acknowledge support from the Institute Strategic Programmes ‘Designing Future Wheat’ (BB/P016855/1), ‘Understanding and Exploiting Plant and Microbial Secondary Metabolism’ (BB/J004596/1) and ‘Molecules from Nature’ (BB/P012523/1) from the UK Biotechnology and Biological Sciences Research Council to the John Innes Centre and the European funded COST ACTION FA1106 QualityFruit. VT, PV and CM have also received funding from the European Union’s Horizon 2020 research and innovation programme through the TomGEM project under grant agreement No. 679796. The funding bodies had no role in the design of the study, collection, analysis and interpretation of data nor in writing the manuscript.Background: Flavonoids are produced in all flowering plants in a wide range of tissues including in berry fruits. These compounds are of considerable interest for their biological activities, health benefits and potential pharmacological applications. However, transcriptomic and genomic resources for wild and cultivated berry fruit species are often limited, despite their value in underpinning the in-depth study of metabolic pathways, fruit ripening as well as in the identification of genotypes rich in bioactive compounds. Results: To access the genetic diversity of wild and cultivated berry fruit species that accumulate high levels of phenolic compounds in their fleshy berry(-like) fruits, we selected 13 species from Europe, South America and Asia representing eight genera, seven families and seven orders within three clades of the kingdom Plantae. RNA from either ripe fruits (ten species) or three ripening stages (two species) as well as leaf RNA (one species) were used to construct, assemble and analyse de novo transcriptomes. The transcriptome sequences are deposited in the BacHBerryGEN database (http://jicbio.nbi.ac.uk/berries) and were used, as a proof of concept, via its BLAST portal (http://jicbio.nbi.ac.uk/berries/blast.html) to identify candidate genes involved in the biosynthesis of phenylpropanoid compounds. Genes encoding regulatory proteins of the anthocyanin biosynthetic pathway (MYB and basic helix-loop-helix (bHLH) transcription factors and WD40 repeat proteins) were isolated using the transcriptomic resources of wild blackberry (Rubus genevieri) and cultivated red raspberry (Rubus idaeus cv. Prestige) and were shown to activate anthocyanin synthesis in Nicotiana benthamiana. Expression patterns of candidate flavonoid gene transcripts were also studied across three fruit developmental stages via the BacHBerryEXP gene expression browser (http://www.bachberryexp.com) in R. genevieri and R. idaeus cv. Prestige. Conclusions: We report a transcriptome resource that includes data for a wide range of berry(-like) fruit species that has been developed for gene identification and functional analysis to assist in berry fruit improvement. These resources will enable investigations of metabolic processes in berries beyond the phenylpropanoid biosynthetic pathway analysed in this study. The RNA-seq data will be useful for studies of berry fruit development and to select wild plant species useful for plant breeding purposes.publishersversionpublishe

BacHBerry: BACterial Hosts for production of Bioactive phenolics from bERRY fruits

BACterial Hosts for production of Bioactive phenolics from bERRY fruits (BacHBerry) was a 3-year project funded by the Seventh Framework Programme (FP7) of the European Union that ran between November 2013 and October 2016. The overall aim of the project was to establish a sustainable and economically-feasible strategy for the production of novel high-value phenolic compounds isolated from berry fruits using bacterial platforms. The project aimed at covering all stages of the discovery and pre-commercialization process, including berry collection, screening and characterization of their bioactive components, identification and functional characterization of the corresponding biosynthetic pathways, and construction of Gram-positive bacterial cell factories producing phenolic compounds. Further activities included optimization of polyphenol extraction methods from bacterial cultures, scale-up of production by fermentation up to pilot scale, as well as societal and economic analyses of the processes. This review article summarizes some of the key findings obtained throughout the duration of the project

Auxin response factor SlARF2 Is an essential component of the regulatory mechanism controlling fruit ripening in tomato

Ethylene is the main regulator of climacteric fruit ripening, by contrast the putative role of other phytohormones in this process remains poorly understood. The present study brings auxin signaling components into the mechanism regulating tomato fruit ripening through the functional characterization of Auxin Response Factor2 (SlARF2) which encodes a downstream component of auxin signaling. Two paralogs, SlARF2A and SlARF2B, are found in the tomato genome, both displaying a marked ripening-associated expression but distinct responsiveness to ethylene and auxin. Down-regulation of either SlARF2A or SlARF2B resulted in ripening defects while simultaneous silencing of both genes led to severe ripening inhibition suggesting a functional redundancy among the two ARFs. Tomato fruits under-expressing SlARF2 produced less climacteric ethylene and exhibited a dramatic down-regulation of the key ripening regulators RIN, CNR, NOR and TAGL1. Ethylene treatment failed to reverse the non-ripening phenotype and the expression of ethylene signaling and biosynthesis genes was strongly altered in SlARF2 down-regulated fruits. Although both SlARF proteins are transcriptional repressors the data indicate they work as positive regulators of tomato fruit ripening. Altogether, the study defines SlARF2 as a new component of the regulatory network controlling the ripening process in tomato

AUXIN RESPONSE FACTOR 2 Intersects Hormonal Signals in the Regulation of Tomato Fruit Ripening.

The involvement of ethylene in fruit ripening is well documented, though knowledge regarding the crosstalk between ethylene and other hormones in ripening is lacking. We discovered that AUXIN RESPONSE FACTOR 2A (ARF2A), a recognized auxin signaling component, functions in the control of ripening. ARF2A expression is ripening regulated and reduced in the rin, nor and nr ripening mutants. It is also responsive to exogenous application of ethylene, auxin and abscisic acid (ABA). Over-expressing ARF2A in tomato resulted in blotchy ripening in which certain fruit regions turn red and possess accelerated ripening. ARF2A over-expressing fruit displayed early ethylene emission and ethylene signaling inhibition delayed their ripening phenotype, suggesting ethylene dependency. Both green and red fruit regions showed the induction of ethylene signaling components and master regulators of ripening. Comprehensive hormone profiling revealed that altered ARF2A expression in fruit significantly modified abscisates, cytokinins and salicylic acid while gibberellic acid and auxin metabolites were unaffected. Silencing of ARF2A further validated these observations as reducing ARF2A expression let to retarded fruit ripening, parthenocarpy and a disturbed hormonal profile. Finally, we show that ARF2A both homodimerizes and interacts with the ABA STRESS RIPENING (ASR1) protein, suggesting that ASR1 might be linking ABA and ethylene-dependent ripening. These results revealed that ARF2A interconnects signals of ethylene and additional hormones to co-ordinate the capacity of fruit tissue to initiate the complex ripening process

Altered ripening phenotypes of <i>SlARF2</i> down-regulated lines.

<p>(A) Ripening phenotypes of <i>SlARF2A-RNAi; SlARF2B-RNAi</i> and <i>SlARF2AB-RNAi</i> fruits at mature green (upper panel) and ripe (lower panel) stages. The <i>SlARF2A/SlARF2B-RNAi</i> fruits show spiky phenotype at mature green stage and ripe stage fruits, <i>SlARF2AB-RNAi</i> mutant displays inhibited ripening. (B) Time (number of days) from anthesis to breaker in wild type and two independent <i>SlARF2AB-RNAi</i> lines. (C) Ripening phenotypes of wild-type (WT) and <i>SlARF2AB-RNAi</i> fruits. Transgenic fruits never reach a full red color. Br = breaker stage; Br+3 = 3 days post-breaker stage; Br+5 = 5 days post-breaker stage; Br+7 = 7 days post-breaker stage. (D) Effect of ethylene treatment on wild type (WT) and <i>SlARF2AB-RNAi</i> fruit. Mature green fruits from WT and <i>SlARF2AB-RNAi</i> lines were treated 2 hours and 3 times per day with 10 ppm ethylene or with air for 3 days. After 7 days, both ethylene treated and untreated wild type fruit reached full red while <i>SlARF2AB-RNAi</i> fruits treated or untreated displayed orange sectors on the fruit surface and never get red.</p

Subcellular localization and functional analysis of SlARF2A and SlARF2B by single cell system.

<p>(A) Subcellular localization of tomato SlARF2A/2B proteins. SlARF2A/2B-GFP fusion proteins were transiently expressed in BY-2 tobacco protoplasts and subcellular localization was analyzed by confocal laser scanning microscopy. The merged pictures of the green fluorescence channel (left panels) and the corresponding bright field (middle panels) are shown in the right panels. The scale bar indicates 10 μm. The top pictures correspond to control cells expressing GFP alone. The middle and bottom pictures correspond to cells expressing the SlARF2A-GFP and SlARF2B-GFP fusion proteins, respectively. (B) SlARF2A/2B protein represses the activity of DR5 <i>in vivo</i>. SlARF2A/2B proteins were challenged with a synthetic auxin-responsive promoter called <i>DR5</i> fused to the GFP reporter gene. A transient expression assay using a single cell system was performed to measure the reporter gene activity. Tobacco protoplasts were transformed either with the reporter construct (DR5::GFP) alone or with both the reporter and effector constructs (35S::SlARF2A/2B) and incubated in the presence or absence of 50 μM 2,4-D. GFP fluorescence was measured 16 h after transfection. For each assay, three biological replicates were performed. GFP mean fluorescence is indicated in arbitrary unit (a.u.) ± standard error.</p

Expression pattern of <i>SlARF2A</i> and <i>SlARF2B</i> in <i>SlARF2 RNAi</i> transgenic lines.

<p>(A) <i>SlARF2A-RNAi</i>, <i>SlARF2B-RNAi</i> and <i>SlARF2AB-RNAi</i> constructs. AB = specific fragment in the DBD binding domain for both SlARF2A and SlARF2B used in <i>SlARF2AB-RNAi</i> construct. A = specific fragment in the middle region of SlARF2A used in <i>SlARF2A-RNAi</i> construct, B = specific fragment in the middle region of SlARF2B used in <i>SlARF2B-RNAi</i> construct. (B) transcript levels of <i>SlARF2A</i> and <i>SlARF2B</i> in <i>RNAi</i> transgenic lines analyzed by quantitative RT-PCR. Expression of <i>SlARF2A</i>/<i>SlARF2B</i> in wild type was taken as reference (relative mRNA level 100%) and the <i>SlActin</i> gene as an internal control. % remaining expression of <i>SlARF2A</i> and <i>SlARF2B</i> transcript levels relative to the reference. Error bars mean ±SD of three biological replicates. Stars indicate a statistical significance (p<0.05) using Student’s t-test. (C) SlARF2A negatively regulates the activity of <i>SlARF2B</i> promoter. Tobacco protoplasts were transformed either with the reporter construct (pSlARF2B::GFP) alone or with both the reporter and effector constructs (35S-SlARF2A) and GFP fluorescence was measured 16 h after transfection. Effector construct lacking SlARF2A was used as control for the co-transfection experiments. Transformations were performed in triplicate. Mean fluorescence is indicated in arbitrary unit (a.u.) ± standard error. Stars indicate a statistical significance (Student’s t-test): * p-value < 0.05, ** p-value < 0.01.</p

The expression of a number of ripening-related genes is altered in <i>SlARF2AB-RNAi</i> plants.

<p>Quantitative RT-PCR relative expression of ripening regulator genes in wild-type (WT) and <i>SlARF2AB-RNAi</i> lines during fruit ripening. Total RNA was extracted from the indicated developmental stages of fruit (breaker, Br; Br+2, 2 days post-breaker; Br+8, 8 days post-breaker). The relative mRNA levels of each gene in WT at the breaker (Br) stage were standardized to 1.0, referring to the <i>SlActin</i> gene as internal control. Error bar means ±SD of three biological replicates. Stars indicate statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01. AP2a, APETALA2/ERF gene; CNR, colorless non-ripening; HB-1, HD-Zip homeobox; NOR, non-ripening; RIN, ripening inhibitor; TAGL1, tomato AGAMOUS-LIKE 1. FUL1, FUL2 MADS domain transcription factors; E4, E8 ethylene-responsive and ripening-regulated genes.</p

The expression of ethylene synthesis and ethylene perception genes is altered in <i>SlARF2AB-RNAi</i> plants.

<p>(A) Expression of ethylene synthesis pathway genes in <i>SlARF2AB-RNAi</i> lines assed by Quantitative RT-PCR. <i>ACO1</i>, <i>ACO2</i>, <i>ACO3</i>, <i>ACO4</i> aminocyclopropane-1-carboxylic acid oxidase; <i>ACS1</i>, <i>ACS2</i>, <i>ACS3</i>, <i>ACS4</i>, <i>ACS6</i> aminocyclopropane-1-carboxylic acid synthases. (B) Expression of ethylene perception genes in <i>SlARF2AB-RNAi</i> assessed by Quantitative RT-PCR. EIN2 ethylene signaling protein; EIL2 and EIL3 are EIN3-like proteins; ETR1, ETR2, ETR3 (NR, never-ripe), ETR4, ETR5, ETR6 ethylene receptors; CTR1 ethylene-responsive protein kinase. ABL1 refers to <i>SlARF2AB-RNAi</i> line 311. Total RNA was extracted from different fruit developmental stages (breaker, Br; Br+2, 2 days post-breaker; Br+8, 8 days post-breaker). The relative mRNA levels of each gene in WT at the breaker (Br) stage were standardized to 1.0, referring to the <i>SlActin</i> gene as internal control. Error bars mean ±SD of three biological replicates. Stars indicate statistical significance using Student’s t-test: * p-value<0.05, ** p-value<0.01.</p

Impact of the down-regulation of <i>SlARF2A</i> and <i>SlARF2B</i> on auxin response assessed <i>in planta</i> following genetic crosses between DR5::GUS and <i>SlARF2</i> down-regulated lines.

<p>(A) Expression pattern of the GUS reporter gene under the control driven by the auxin-inducible DR5 promoter in wild type (WT) and <i>SlARF2</i> down-regulated genetic background. Seedlings were treated with auxin (IAA 20 μM for 3 hours) or with a mock solution. Upper panel: <i>in planta</i> expression of the GUS reporter gene driven by DR5 in WT genetic background in the absence (left) or presence (right) of auxin treatment. Bottom panel: Expression of the GUS reporter gene driven by DR5 in <i>ARF2A RNAi</i> (left), <i>ARF2B RNAi</i> (middle) and <i>ARF2AB RNAi</i> (right) genetic background. (B) Quantitative RT-PCR expression analysis of <i>GUS</i> and <i>SlARF2A/2B</i> genes in WT and <i>SlARF2A</i> and <i>SlARF2B-RNAi</i> lines crossed with DR5::GUS lines. The relative mRNA levels of <i>GUS-1/GUS-2</i> (Upper panel) and <i>SlARF2A/2B</i> (bottom panel) in wild type were standardized to 1.0, referring to the <i>SlActin</i> gene as internal control. Error bars mean ±SD of three biological replicates. *0.01 < p-value < 0.05. DR5-WT = DR5::GUS/WT; DR5-2A = DR5::GUS/ARF2A RNAi; DR5-2B = DR5::GUS/ARF2B RNAi; DR5-2AB = DR5::GUS/ARF2AB RNAi. <i>GUS-1</i> and <i>GUS-2</i> refer to the use of two distinct pairs of primers designed in two distinct regions of the GUS mRNA sequence.</p