Identification of Key Residues for pH Dependent Activation of Violaxanthin De-Epoxidase from Arabidopsis thaliana

Plants are often exposed to saturating light conditions, which can lead to oxidative stress. The carotenoid zeaxanthin, synthesized from violaxanthin by Violaxanthin De-Epoxidase (VDE) plays a major role in the protection from excess illumination. VDE activation is triggered by a pH reduction in the thylakoids lumen occurring under saturating light. In this work the mechanism of the VDE activation was investigated on a molecular level using multi conformer continuum electrostatic calculations, site directed mutagenesis and molecular dynamics. The pKa values of residues of the inactive VDE were determined to identify target residues that could be implicated in the activation. Five such target residues were investigated closer by site directed mutagenesis, whereas variants in four residues (D98, D117, H168 and D206) caused a reduction in enzymatic activity indicating a role in the activation of VDE while D86 mutants did not show any alteration. The analysis of the VDE sequence showed that the four putative activation residues are all conserved in plants but not in diatoms, explaining why VDE in these algae is already activated at higher pH. Molecular dynamics showed that the VDE structure was coherent at pH 7 with a low amount of water penetrating the hydrophobic barrel. Simulations carried out with the candidate residues locked into their protonated state showed instead an increased amount of water penetrating the barrel and the rupture of the H121–Y214 hydrogen bond at the end of the barrel, which is essential for VDE activation. These results suggest that VDE activation relies on a robust and redundant network, in which the four residues identified in this study play a major role

Silencing of the Violaxanthin De-Epoxidase Gene in the Diatom Phaeodactylum tricornutum Reduces Diatoxanthin Synthesis and Non-Photochemical Quenching

Diatoms are a major group of primary producers ubiquitous in all aquatic ecosystems. To protect themselves from photooxidative damage in a fluctuating light climate potentially punctuated with regular excess light exposures, diatoms have developed several photoprotective mechanisms. The xanthophyll cycle (XC) dependent non-photochemical chlorophyll fluorescence quenching (NPQ) is one of the most important photoprotective processes that rapidly regulate photosynthesis in diatoms. NPQ depends on the conversion of diadinoxanthin (DD) into diatoxanthin (DT) by the violaxanthin de-epoxidase (VDE), also called DD de-epoxidase (DDE). To study the role of DDE in controlling NPQ, we generated transformants of P. tricornutum in which the gene (Vde/Dde) encoding for DDE was silenced. RNA interference was induced by genetic transformation of the cells with plasmids containing either short (198 bp) or long (523 bp) antisense (AS) fragments or, alternatively, with a plasmid mediating the expression of a self-complementary hairpin-like construct (inverted repeat, IR). The silencing approaches generated diatom transformants with a phenotype clearly distinguishable from wildtype (WT) cells, i.e. a lower degree as well as slower kinetics of both DD de-epoxidation and NPQ induction. Real-time PCR based quantification of Dde transcripts revealed differences in transcript levels between AS transformants and WT cells but also between AS and IR transformants, suggesting the possible presence of two different gene silencing mediating mechanisms. This was confirmed by the differential effect of the light intensity on the respective silencing efficiency of both types of transformants. The characterization of the transformants strengthened some of the specific features of the XC and NPQ and confirmed the most recent mechanistic model of the DT/NPQ relationship in diatoms

Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis

The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11103-015-0363-5) contains supplementary material, which is available to authorized users

VIGS: A Tool to Study Fruit Development in Solanum lycopersicum

[EN] A visually traceable system for fast analysis of gene functions based on Fruit-VIGS methodology is described. In our system, the anthocyanin accumulation from purple transgenic tomato lines provides the appropriate background for fruit-specific gene silencing. The tomato Del/Ros1 background ectopically express Delila (Del) and Rosea1 (Ros1) transgenes under the control of fruit ripening E8 promoter, activating specifically anthocyanin biosynthesis during tomato fruit ripening. The Virus-Induced Gene Silencing (VIGS) of Delila and Rosea1 produces a color change in the silenced area easily identifiable. Del/Ros1 VIGS is achieved by agroinjection of an infective clone of Tobacco Rattle Virus (pTRV1 and pTRV2 binary plasmids) directly into the tomato fruit. The infective clone contains a small fragment of Del and Ros1 coding regions (named DR module). The co-silencing of reporter Del/Ros1 genes and a gene of interest (GOI) in the same region enables us to identify the precise region where silencing is occurring. The function of the GOI is established by comparing silenced sectors of fruits where both GOI and reporter DR genes have been silenced with fruits in which only the reporter DR genes have been silenced. The Gateway vector pTRV2_DR_GW was developed to facilitate the cloning of different GOIs together with DR genes. Our tool is particularly useful to study genes involved in metabolic processes during fruit ripening, which by themselves would not produce a visual phenotype.We are grateful to Prof. Dinesh Kumar who kindly provided us with TRV-based silencing vectors pTRV1 and pTRV2. We appreciate Prof. Cathie Martin for providing Del/Ros1 transgenic tomato lines. The work described here was supported by BIO2008-034034 grant from the Spanish Ministry of Science and Technology, FPU fellowship from Spanish MICINN and EUSOL project from EU.Fernández Moreno, JP.; Orzáez Calatayud, DV.; Granell Richart, A. (2013). VIGS a tool to study fruit development in Solanum lycopersicum. Virus-Induced Gene Silencing: Methods and Protocols. 975:183-196. https://doi.org/10.1007/978-1-62703-278-0_14S183196975Ratcliff F, Martin-Hernandez AM, Baulcombe DC (2001) Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25:237–245Lu R, Martin-Hernandez AM, Peart JR et al (2003) Virus-induced gene silencing in plants. Methods (San Diego, Calif) 30:296–303Fu DQ, Zhu BZ, Zhu HL et al (2005) Virus-induced gene silencing in tomato fruit. Plant J 43:299–308Shao Y, Zhu HL, Tian HQ, Wang XG et al (2008) Virus-Induce Gene Silencing in Plant Species. Rus J Plant Physiol 55:168–174Burch-Smith TM, Anderson JC, Martin GB et al (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39:734–746Orzaez D, Medina A, Torre S et al (2009) A visual reporter system for virus-induced gene silencing in tomato fruit based on anthocyanin accumulation. Plant Physiol 150:1122–1134Unver T, Budak H (2009) Virus-induced gene silencing, a post transcriptional gene silencing method. Int J Plant Genomic 2009:198–680Orzaez D, Mirabel S, Wieland WH et al (2006) Agroinjection of tomato fruits. A tool for rapid functional analysis of transgenes directly in fruit. Plant physiol 140:3–11Orzaez D, Granell A (2009) Reverse genetics and transient gene expression in fleshy fruits: overcoming plant stable transformation. Plant Signal Behav 4:864–867Ballester AR, Molthoff J, de Vos R et al (2010) Biochemical and molecular analysis of pink tomatoes: deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit color. Plant Physiol 152:71–84Liu Y, Schiff M, Dinesh-Kumar SP (2002) Virus-induced gene silencing in tomato. Plant J 31:777–786Butelli E, Titta L, Giorgio M et al (2008) Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors. Nat Biotechnol 26:1301–1308Davies KM, Marshall GB, Marie Bradley J, Schwinn KE et al (2006) Characterisation of aurone biosynthesis in Antirrhinum majus. Physiol Plant 128:593–603Marti C, Orzaez D, Ellul P et al (2007) Silencing of DELLA induces facultative parthenocarpy in tomato fruits. Plant J 52:865–876Bugos RC, Chiang VL, Zhang XH, Campbell ER et al (1995) RNA isolation from plant tissues recalcitrant to extraction in guanidine. Biotechniques 19:734–744Estornell LH, Orzaez D, Lopez-Pena L et al (2009) A multisite gateway-based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits. Plant Biotechnol J 7:298–30