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 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

Microarray analysis of <i>ARF2-OX</i> fruit.

<p>Gene expression was analyzed in WT at 42 dpa (mature green stage; WT-42G) and 53 dpa (red stage; WT-53R) and green (<i>ARF2-OX</i>-42G) and red (<i>ARF2-OX</i>-42R) patches from <i>ARF2-OX</i> fruit at 42 dpa by microarray analysis. Results are displayed as (A) a principal component analysis (PCA) and (B) a Venn diagram of the differentially expressed genes in the comparisons: <i>ARF2-OX</i>-42G to WT-42G; <i>ARF2-OX</i>-42R to WT-42G; and WT-53R to WT-42G. P-value<0.01 and FDR<0.05; dpa: days post anthesis.</p

Dimerization of the ARF2A protein and its interaction.

<p>ARF2A was cloned downstream of the DNA-binding domain (DB-ARF2A) and co-transformed into yeast with either (A) ARF2A cloned downstream of the activation domain (AD-ARF2A); or (B) ASR1 cloned downstream of the activation domain (AD-ASR1), yeast growth on media lacking leucine, tryptophan, histidine and adenine indicated positive protein-protein interactions. (C) Relative expression levels of <i>ASR1</i> in WT cv. MicroTom fruit at five developmental stages (IG: immature green; MG: mature green; Br: breaker; Or: orange; and R: red), error bars represent SE; statistical significance was evaluated using an ANOVA test (JMP software, SAS) with three biological repeats based on the average of three technical replicates, values indicated by the same letter (a,b,c) are not statistically significant, p-value<0.05. (D) A Bimolecular Fluorescence Complementation assay (BiFC) was carried out by transient expression in tobacco leaves; ARF2A was cloned downstream of the amino-terminal region of YFP (yellow fluorescent protein; YN-ARF2A) and ASR1 was cloned downstream of the carboxy-terminal region of YFP (YC-ASR1); leaf regions were examined for fluorescent signal by light and confocal fluorescence microscopy. Inset zoom region shows that the ARF2A-ASR1 interaction is nuclear localized. Scale bars in the light and confocal fluorescence microscopy represent 50 μm and 10 μm, respectively.</p

Hormone profiling in <i>ARF2-OX</i> transgenic fruit.

<p>WT and <i>ARF2-OX</i> fruit, at 39, 42 and 53 dpa were analyzed for levels of (A) salicylic acid; (B) abscisic acid; (C-G) cytokinins of the <i>trans</i>-zeatin biosynthesis branch and (H-J) cytokinins of the <i>cis</i>-zeatin biosynthesis branch, using UPLC-ESI-MS/MS. Relative expression level of the cytokinin-related genes (K) <i>CKX7-like</i>; (L) <i>LOG8-like</i> was analysed by qRT-PCR. Black bars represent WT, hatched bars <i>ARF2-OX</i> at 39 dpa, white bars <i>ARF2-OX</i> green patches at 42 and 53 dpa and grey bars <i>ARF2-OX</i> red patches at 42 and 53 dpa. Error bars represent SE. Statistical significance was evaluated using a student’s t-test, *p-value<0.05 and **p-value<0.01; dpa: days post anthesis.</p

Over-expression of <i>ARF2A</i> in transgenic tomato plants.

<p>(A) Silent mutations in the tasi-RNA recognition site introduced into the <i>ARF2A</i> open reading frame; (B) days from anthesis to breaker stage in WT and <i>ARF2-OX</i> fruit (Error bars represent SE); (C) blotchy ripening phenotype observed in <i>ARF2-OX</i> fruit (DPA: days post anthesis); and (D) blotchy ripening occasionally observed in WT fruit which eventually all turn red in comparison with the <i>ARF2-OX</i> lines which have regions which remain yellow; (E) A scheme representing the sampling of tissues from WT and <i>ARF2-OX</i> fruit at 39, 42 and 53 dpa, in this study; when patches were visible at 42 and 53 dpa, they were harvested and treated separately. Relative expression levels of <i>ARF2A</i> variants were analyzed by qRT-PCR in WT and patches of <i>ARF2-OX</i> fruit at 39 and 42 dpa, using oligonucleotides specific to (F) the transgene (<i>trans-ARF2A</i>); and (G) the endogenous <i>ARF2A</i> gene (<i>endo-ARF2A</i>). Black bars represent WT, hatched bars <i>ARF2-OX</i> at 39 dpa, white bars <i>ARF2-OX</i> green patches at 42 dpa and grey bars <i>ARF2-OX</i> red patches at 42 dpa. Error bars represent SE. DPH: days post-harvest; n.d.: not detected; dpa: days post anthesis.</p

Phenotype and expression levels of <i>ARF2</i> genes in <i>ARF2as</i> transgenic lines.

<p>Fruit of <i>ARF2as</i> lines were analysed for relative expression levels by qRT-PCR of (A) <i>ARF2A;</i> and (B) <i>ARF2B</i>. (C-D) The ripening of <i>ARF2as</i> fruit is delayed as compared to WT. (D-E) <i>ARF2as</i> fruit were parthenocarpic or nearly parthenocarpic with reduced number of seeds. (F) Principle component analysis (PCA) plot from untargeted analysis of metabolites. DPA: days post anthesis; error bars represent SE; Statistical significance was evaluated using a student’s t-test, **p-value<0.01.</p

<i>ARF2A</i> expression in tomato fruit.

<p>Relative expression levels of <i>ARF2A</i> analyzed by qRT-PCR, in (A) WT fruit at five developmental stages; (B) <i>rin</i> and <i>nor</i> mutants; (C) <i>TAGL1</i> over-expressing fruit (<i>35S</i>:<i>TAGL1</i>); and (D) <i>nr</i> mutant. Relative expression levels of <i>ARF2A</i> in fruit at three developmental stages, treated with (E) 1-MCP; (F) ethylene; (H) NAA; and (J) ABA. Relative expression levels of <i>ARF2A</i> in fruit at the MG stage, at 0, 2, 4 and 6 days post-treatment with (G) ethrel; (I) NAA; and (K) ABA. Error bars represent SE. Statistical significance was evaluated using a student’s t-test, *p-value<0.05, **p-value<0.01 and ***p-value<0.001; dpa: days post anthesis; IG: immature green; MG: mature green; Br: breaker; Or: orange; and R: red.</p

Metabolic analysis of <i>ARF2-OX</i> fruit.

<p>WT and <i>ARF2-OX</i> fruit were analyzed at 42 and 53 dpa by UPLC-qTOF-MS in positive mode, (A) results are visualized by a principle component analysis (PCA) plot; and displayed as histograms for (B) targeted flavonoids (upper row), and targeted glycoalkaloids (lower row). (C) Isoprenoids were analyzed in WT and <i>ARF2-OX</i> fruit at 42 and 53 dpa by HPLC. Grey bars represent WT, black bars <i>ARF2-OX</i> green/yellow patches and white bars <i>ARF2-OX</i> red patches. Error bars represent SE. Statistical significance was evaluated using a student’s t-test, *p-value<0.05; dpa: days post anthesis.</p

Altering ethylene signaling in <i>ARF2-OX</i> transgenic fruit.

<p><i>ARF2-OX</i> fruit at the mature green (MG) stage, before the visual appearance of patches, were treated with either (A) ethrel, or (B) 1-MCP, and phenotypes were observed at (A) 10 and 16, or (B) 7 and 10 DPT. (C) Ethylene emission was measured from WT and <i>ARF2-OX</i> fruit harvested at the MG stage, every 1–3 days for 16 days, the red bars and arrows indicate the breaker stage. Error bars represent SE. DPT: days post treatment.</p