Eye movement desensitization and processing for adolescents with major depressive disorder: study protocol for a multi-site randomized controlled trial.

BACKGROUND: Major depressive disorder (MDD) is one of the most common mental disorders in adolescence carrying a serious risk of adverse development later in life. Extant treatments are limited in effectiveness and have high drop-out and relapse rates. A body of literature has been published on the association between distressing/ traumatic experiences and development and maintenance of MDD, but the effectiveness of a trauma-focused treatment approach for MDD has hardly been studied. This study aims to determine the effectiveness of eye movement desensitization and reprocessing (EMDR) therapy as stand-alone intervention in adolescents diagnosed with MDD. METHODS: This study will be a randomized controlled trial with two conditions: (1) EMDR treatment (6 sessions) and (2) waiting list condition (WL: 6 weeks, followed by EMDR treatment). First, participants receive a baseline measure after which they will be randomized. Participants will be assessed post-intervention after which the WL participants will also receive six EMDR sessions. Follow-up assessments will be conducted at 3 and 6 months follow-up. STUDY POPULATION: In total, 64 adolescents (aged 12-18) diagnosed with a major depressive disorder (DSM-5) and identified memories of at least one distressing or traumatic event related to the depressive symptomatology will be included. Main study parameters/endpoints: Primary outcome variables will be the percentage of patients meeting criteria for MDD classification, and level of depressive symptoms. Secondary outcome measures include symptoms of PTSD, anxiety, and general social-emotional problems. At baseline, family functioning and having experienced emotional abuse or neglect will be assessed to explore whether these factors predict post-treatment outcome. DISCUSSION: With the present study, we aim to investigate whether EMDR as a trauma-focussed brief intervention may be effective for adolescents with a primary diagnosis of MDD. EMDR has been proven an effective treatment for traumatic memories in other disorders. It is hypothesized that traumatic memories play a role in the onset and maintenance of depressive disorders. Particularly in adolescence, early treatment of these traumatic memories is warranted to prevent a more chronic or recurrent course of the disorder. TRIAL REGISTRATION: International Clinical Trial Registry Platform (ICTRP): NL9008 (30-10-2020).

Pleiotropic effects of the Arabidopsis cryptochrome 2 allelic variation underlie fruit trait-related QTL.

The previous molecular identification of a flowering time QTL segregating in the Arabidopsis L er x Cvi cross, demonstrated that natural allelic variation at the blue light photoreceptor CRY2 gene affects flowering time (El-Assal et al., 2001). In addition, previous works on the same cross have mapped several QTL affecting other unrelated life history traits in the CRY2 genomic region. In the present report, we have used a set of Arabidopsis L er transgenic plants carrying four different functional CRY2 transgenes for phenotypic analyses, with the aim of exploring the extent of pleiotropy of CRY2 allelic variation. It is concluded that previously identified QTL affecting fruit length, ovule number per fruit, and percentage of unfertilized ovules are caused by this same Ler/Cvi CRY2 allelic variation. In addition, dose effects of the CRY2-L er allele are detected for fruit length. A seed weight QTL at the map position of CRY2 could not be confirmed and also no effect on seed dormancy was observed. Thus, it is shown that transgenic plants carrying different alleles can be a useful tool to attribute QTL for different complex traits to a specific locus, even when the relationship among the traits has not been previously suggested.

The genetics of phytate and phosphate accumulation in seeds and leaves of Arabidopsis thaliana, using natural variation.

Phytate (myo-inositol-1,2,3,4,5,6-hexakisphosphate, InsP6) is the most abundant P-containing compound in plants, and an important anti-nutritional factor, due to its ability to complex essential micro-nutrients, e.g. iron and zinc. Analysis of natural variation for InsP6 and Pi accumulation in seeds and leaves for a large number of accessions of Arabidopsis thaliana, using a novel method for InsP6 detection, revealed a wide range of variation in InsP6 and Pi levels, varying from 7.0 mg to 23.1 mg of InsP6 per gram of seed. Quantitative trait locus (QTL) analysis of InsP6 and Pi levels in seeds and leaves, using an existing recombinant inbred line population, was performed in order to identify a gene(s) that is (are) involved in the regulation of InsP6 accumulation. Five genomic regions affecting the quantity of the InsP6 and Pi in seeds and leaves were identified. One of them, located on top of chromosome 3, affects all four traits. This QTL appears as the major locus responsible for the observed variation in InsP6 and Pi contents in the L er/Cvi RIL population; the L er allele decreases the content of both InsP6 and Pi in seeds and in leaves. The InsP6/Pi locus was further fine-mapped to a 99-kb region, containing 13 open reading frames. The maternal inheritance of the QTL and the positive correlation between InsP6 and total Pi levels both in seeds and in leaves indicate that the difference in InsP6 level between L er and Cvi is likely to be caused by a difference in transport rather than by an alteration in the biosynthesis. Therefore, we consider the vacuolar membrane ATPase subunit G, located in the region of interest, as the most likely candidate gene for InsP6/Pi.

A QTL for flowering time in Arabidopsis reveals a novel allele of CRY2.

Variation of flowering time is found in the natural populations of many plant species. The underlying genetic variation, mostly of a quantitative nature, is presumed to reflect adaptations to different environments contributing to reproductive success. Analysis of natural variation for flowering time in Arabidopsis thaliana has identified several quantitative trait loci (QTL), which have yet to be characterized at the molecular level. A major environmental factor that determines flowering time is photoperiod or day length, the length of the light period, which changes across the year differently with geographical latitude. We identified the EDI locus as a QTL partly accounting for the difference in flowering response to the photoperiod between two Arabidopsis accessions: the laboratory strain Landsberg erecta (Ler), originating in Northern Europe, and Cvi, collected in the tropical Cape Verde Islands. Positional cloning of the EDI QTL showed it to be a novel allele of CRY2, encoding the blue-light photoreceptor cryptochrome-2 that has previously been shown to promote flowering in long-day (LD) photoperiods. We show that the unique EDI flowering phenotype results from a single amino-acid substitution that reduces the light-induced downregulation of CRY2 in plants grown under short photoperiods, leading to early flowering.

The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium.

Phenolic compounds that are present in the testa interfere with the physiology of seed dormancy and germination. We isolated a recessive Arabidopsis mutant with pale brown seeds, transparent testa12 (tt12), from a reduced seed dormancy screen. Microscopic analysis of tt12 developing and mature testas revealed a strong reduction of proanthocyanidin deposition in vacuoles of endothelial cells. Double mutants with tt12 and other testa pigmentation mutants were constructed, and their phenotypes confirmed that tt12 was affected at the level of the flavonoid biosynthetic pathway. The TT12 gene was cloned and found to encode a protein with similarity to prokaryotic and eukaryotic secondary transporters with 12 transmembrane segments, belonging to the MATE (multidrug and toxic compound extrusion) family. TT12 is expressed specifically in ovules and developing seeds. In situ hybridization localized its transcript in the endothelium layer, as expected from the effect of the tt12 mutation on testa flavonoid pigmentation. The phenotype of the mutant and the nature of the gene suggest that TT12 may control the vacuolar sequestration of flavonoids in the seed coat endothelium.

Genetic dissection of blue-light sensing in tomato using mutants deficient in cryptochrome 1 and phytochromes A, B1 and B2.

Several novel allelic groups of tomato (Solanum lycopersicum L.) mutants with impaired photomorphogenesis have been identified after gamma-ray mutagenesis of phyA phyB1 double-mutant seed. Recessive mutants in one allelic group are characterized by retarded hook opening, increased hypocotyl elongation and reduced hypocotyl chlorophyll content under white light (WL). These mutants showed a specific impairment in response to blue light (BL) resulting from lesions in the gene encoding the BL receptor cryptochrome 1 (cry1). Phytochrome A and cry1 are identified as the major photoreceptors mediating BL-induced de-etiolation in tomato, and act under low and high irradiances, respectively. Phytochromes B1 and B2 also contribute to BL sensing, and the relative contribution of each of these four photoreceptors differs according to the light conditions and the specific process examined. Development of the phyA phyB1 phyB2 cry1 quadruple mutant under WL is severely impaired, and seedlings die before flowering. The quadruple mutant is essentially blind to BL, but experiments employing simultaneous irradiation with BL and red light suggest that an additional non-phytochrome photoreceptor may be active under short daily BL exposures. In addition to effects on de-etiolation, cry1 is active in older, WL-grown plants, and influences stem elongation, apical dominance, and the chlorophyll content of leaves and fruit. These results provide the first mutant-based characterization of cry1 in a plant species other than Arabidopsis.

Cell division activity during apical hook development.

Growth during plant development is predominantly governed by the combined activities of cell division and cell elongation. The relative contribution of both activities controls the growth of a tissue. A fast change in growth is exhibited at the apical hypocotyl of etiolated seedlings where cells grow at different rates to form a hook-like structure, which is traditionally assumed to result from differential cell elongation. Using new tools we show asymmetric distribution of cell division during early stages of hook development. Cell divisions in the apical hook were predominantly found in subepidermal layers during an early step of hook development, but were absent in mutants exhibiting a hookless phenotype. In addition, during exaggeration of hook curvature, which is mediated by ethylene, a rapid change in the combined activities of cell division and cell elongation was detected. Our results indicate a fast change in cell division activity during apical hook development. We suggest that cell division together with cell elongation contributes to apical hook growth. Our results emphasize the change in the relative contribution of cell division and cell elongation in a fast growing structure like the apical hook.

Sequential steps for developmental arrest in Arabidopsis seeds.

The continuous growth of the plant embryo is interrupted during the seed maturation processes which results in a dormant seed. The embryo continues development after germination when it grows into a seedling. The embryo growth phase starts after morphogenesis and ends when the embryo fills the seed sac. Very little is known about the processes regulating this phase. We describe mutants that affect embryo growth in two sequential developmental stages. Firstly, embryo growth arrest is regulated by the FUS3/LEC type genes, as mutations in these genes cause a continuation of growth in immature embryos. Secondly, a later stage of embryo dormancy is regulated by ABI3 and abscisic acid; abi3 and aba1 mutants exhibit premature germination only after embryos mature. Mutations affecting both developmental stages result in an additive phenotype and double mutants are highly viviparous. Embryo growth arrest is regulated by cell division activities in both the embryo and the endosperm, which are gradually switched off at the mature embryo stage. In the fus3/lec mutants, however, cell division in both the embryo and endosperm is not arrested, but rather is prolonged throughout seed maturation. Furthermore ectopic cell division occurs in seedlings. Our results indicate that seed dormancy is secured via at least two sequential developmental processes: embryo growth arrest, which is regulated by cell division and embryo dormancy.

Genetic analysis of seed-soluble oligosaccharides in relation to seed storability of Arabidopsis.

Seed oligosaccharides (OSs) and especially raffinose series OSs (RSOs) are hypothesized to play an important role in the acquisition of desiccation tolerance and consequently in seed storability. In the present work we analyzed the seed-soluble OS (sucrose, raffinose, and stachyose) content of several Arabidopsis accessions and thus identified the genotype Cape Verde Islands having a very low RSO content. By performing quantitative trait loci (QTL) mapping in a recombinant inbred line population, we found one major QTL responsible for the practically monogenic segregation of seed stachyose content. This locus also affected the content of the two other OSs, sucrose, and raffinose. Two candidate genes encoding respectively for galactinol synthase and raffinose synthase were located within the genomic region around this major QTL. In addition, three smaller-effect QTL were identified, each one specifically affecting the content of an individual OS. Seed storability was analyzed in the same recombinant inbred line population by measuring viability (germination) under two different seed aging assays: after natural aging during 4 years of dry storage at room temperature and after artificial aging induced by a controlled deterioration test. Thus, four QTL responsible for the variation of this trait were mapped. Comparison of the QTL genetic positions showed that the genomic region containing the major OS locus did not significantly affect the seed storability. We concluded that in the studied material neither RSOs nor sucrose content had a specific effect on seed storability.

The late flowering phenotype of fwa mutants is caused by gain-of-function epigenetic alleles of a homeodomain gene.

The transition to flowering in Arabidopsis thaliana is delayed in fwa mutant plants. FWA was identified by loss-of-function mutations in normally flowering revertants of the fwa mutant, and it encodes a homeodomain-containing transcription factor. The DNA sequence of wild-type and fwa mutant alleles was identical in the genomic region of FWA. Furthermore, the FWA gene is ectopically expressed in fwa mutants and silenced in mature wild-type plants. This silencing is associated with extensive methylation of two direct repeats in the 5' region of the gene. The late flowering phenotype, ectopic FWA expression, and hypomethylation of the repeats were also induced in the ddm1 hypomethylated background. Mechanisms for establishment and maintenance of the epigenetic mark on FWA are discussed.

Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato.

The role of phytochrome B2 (phyB2) in the control of photomorphogenesis in tomato (Solanum lycopersicum L.) has been investigated using recently isolated mutants carrying lesions in the PHYB2 gene. The physiological interactions of phytochrome A (phyA), phytochrome B1 (phyB1) and phyB2 have also been explored, using an isogenic series of all possible mutant combinations and several different phenotypic characteristics. The loss of phyB2 had a negligible effect on the development of white-light-grown wild-type or phyA-deficient plants, but substantially enhanced the elongated pale phenotype of the phyB1 mutant. This redundancy was also seen in the control of de-etiolation under continuous red light (R), where the loss of phyB2 had no detectable effect in the presence of phyB1. Under continuous R, phyA action was largely independent of phyB1 and phyB2 in terms of the control of hypocotyl elongation, but antagonized the effects of phyB1 in the control of anthocyanin synthesis, indicating that photoreceptors may interact differently to control different traits. Irradiance response curves for anthocyanin synthesis revealed that phyB1 and phyB2 together mediate all the detectable response to high-irradiance R, and, surprisingly, that the phyA-dependent low-irradiance component is also strongly reduced in the phyB1 phyB2 double mutant. This is not associated with a reduction in phyA protein content or responsiveness to continuous far-red light (FR), suggesting that phyB1 and phyB2 specifically influence phyA activity under low-irradiance R. Finally, the phyA phyB1 phyB2 triple mutant showed strong residual responsiveness to supplementary daytime FR, indicating that at least one of the two remaining phytochromes plays a significant role in tomato photomorphogenesis.

The Arabidopsis aldehyde oxidase 3 (AAO3) gene product catalyzes the final step in abscisic acid biosynthesis in leaves.

Abscisic acid (ABA) is a plant hormone involved in seed development and germination and in responses to various environmental stresses. The last step of ABA biosynthesis involves oxidation of abscisic aldehyde, and aldehyde oxidase (EC ) is thought to catalyze this reaction. An aldehyde oxidase isoform, AOdelta, encoded by AAO3, one of four Arabidopsis aldehyde oxidase genes (AAO1, AAO2, AAO3, and AAO4), is the most likely candidate for the enzyme, because it can efficiently catalyze the oxidation of abscisic aldehyde to ABA. Here, we report the isolation and characterization of an ABA-deficient Arabidopsis mutant that maps at the AAO3 locus. The mutant exhibits a wilty phenotype in rosette leaves, but seed dormancy is not affected. ABA levels were significantly reduced in the mutant leaves, explaining the wilty phenotype in rosettes, whereas the level in the mutant seeds was less reduced. No AOdelta activity could be detected in the rosette leaves of the mutant. Sequence data showed that the mutant contains a G to A substitution in the AAO3 gene. The mutation causes incorrect splicing of the ninth intron of AAO3 mRNA. We thus conclude that the ABA-deficient mutant is impaired in the AAO3 gene and that the gene product, AOdelta, is an aldehyde oxidase that catalyzes the last step of ABA biosynthesis in Arabidopsis, specifically in rosette leaves. Other aldehyde oxidases may be involved in ABA biosynthesis in other organs.

Influence of the testa on seed dormancy, germination, and longevity in Arabidopsis.

The testa of higher plant seeds protects the embryo against adverse environmental conditions. Its role is assumed mainly by controlling germination through dormancy imposition and by limiting the detrimental activity of physical and biological agents during seed storage. To analyze the function of the testa in the model plant Arabidopsis, we compared mutants affected in testa pigmentation and/or structure for dormancy, germination, and storability. The seeds of most mutants exhibited reduced dormancy. Moreover, unlike wild-type testas, mutant testas were permeable to tetrazolium salts. These altered dormancy and tetrazolium uptake properties were related to defects in the pigmentation of the endothelium and its neighboring crushed parenchymatic layers, as determined by vanillin staining and microscopic observations. Structural aberrations such as missing layers or a modified epidermal layer in specific mutants also affected dormancy levels and permeability to tetrazolium. Both structural and pigmentation mutants deteriorated faster than the wild types during natural aging at room temperature, with structural mutants being the most strongly affected.

Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid.

The mechanisms imposing a gibberellin (GA) requirement to promote the germination of dormant and non-dormant Arabidopsis seeds were analyzed using the GA-deficient mutant ga1, several seed coat pigmentation and structure mutants, and the abscisic acid (ABA)-deficient mutant aba1. Testa mutants, which exhibit reduced seed dormancy, were not resistant to GA biosynthesis inhibitors such as tetcyclacis and paclobutrazol, contrarily to what was found before for other non-dormant mutants in Arabidopsis. However, testa mutants were more sensitive to exogenous GAs than the wild-types in the presence of the inhibitors or when transferred to a GA-deficient background. The germination capacity of the ga1-1 mutant could be integrally restored, without the help of exogenous GAs, by removing the envelopes or by transferring the mutation to a tt background (tt4 and ttg1). The double mutants still required light and chilling for dormancy breaking, which may indicate that both agents can have an effect independently of GA biosynthesis. The ABA biosynthesis inhibitor norflurazon was partially efficient in releasing the dormancy of wild-type and mutant seeds. These results suggest that GAs are required to overcome the germination constraints imposed both by the seed coat and ABA-related embryo dormancy.

Naturally occurring variation in Arabidopsis: an underexploited resource for plant genetics.

The definition of gene functions requires the phenotypic characterization of genetic variants. Currently, such functional analysis of Arabidopsis genes is based largely on laboratory-induced mutants that are selected in forward and reverse genetic studies. An alternative complementary source of genetic variation is available: the naturally occurring variation among accessions. The multigenic nature of most of this variation has limited its application until now. However, the use of genetic methods developed to map quantitative trait loci, in combination with the characteristics and resources available for molecular biology in Arabidopsis, allow this variation to be exploited up to the molecular level. Here, we describe the current tools available for the forward genetic analysis of this variation, and review the recent progress in the detection and mapping of loci and the cloning of large-effect genes.

Natural allelic variation identifies new genes in the Arabidopsis circadian system.

We have analysed the circadian rhythm of Arabidopsis thaliana leaf movements in the accession Cvi from the Cape Verde Islands, and in the commonly used laboratory strains Columbia (Col) and Landsberg (erecta) (Ler), which originated in Northern Europe. The parental lines have similar rhythmic periods, but the progeny of crosses among them reveal extensive variation for this trait. An analysis of 48 Ler/Cvi recombinant inbred lines (RILs) and a further 30 Ler/Col RILs allowed us to locate four putative quantitative trait loci (QTLs) that control the period of the circadian clock. Near-isogenic lines (NILs) that contain a QTL in a small, defined chromo- somal region allowed us to confirm the phenotypic effect and to map the positions of three period QTLs, designated ESPRESSO, NON TROPPO and RALENTANDO. Quantitative trait loci at the locations of RALENTANDO and of a fourth QTL, ANDANTE, were identified in both Ler/Cvi and Ler/Col RIL populations. Some QTLs for circadian period are closely linked to loci that control flowering time, including FLC. We show that flc mutations shorten the circadian period such that the known allelic variation in the MADS-box gene FLC can account for the ANDANTE QTL. The QTLs ESPRESSO and RALENTANDO identify new genes that regulate the Arabidopsis circadian system in nature, one of which may be the flowering-time gene GIGANTEA.

Importance of the B2 domain of the Arabidopsis ABI3 protein for Em and 2S albumin gene regulation.

Genetic and molecular studies have shown that the Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3) protein plays a prominent role in the control of seed maturation. The ABI3 protein and its orthologues from various other plant species share four domains of high sequence identity, including three basic domains designated as B1, B2 and B3. The leaky abi3-1 mutation is a single amino acid substitution within the B3 domain. A new abi3 allele, abi3-7, was generated by mutagenizing abi3-1 seeds. The abi3-7 line contains, in addition to the abi3-1 mutation, a point mutation that converts residue Ala-458 into Thr within the B2 domain of the ABI3 protein. This Ala residue is absolutely conserved in all known ABI3 orthologues. Abi3-7 seeds display reductions in dormancy and in sensitivity to abscisic acid which are intermediate between those of the leaky abi3-1 and of the severe abi3-4 and abi3-5 mutants. Accumulation and distribution of At2S1 and At2S2 albumin mRNA as well as of AtEm1 and AtEm6 late embryogenesis-abundant proteins and mRNA have been analyzed. Both At2S1 and At2S2 mRNA are reduced in abi3-7, but distribution of At2S2 is spatially restricted. Accumulation of AtEm6 protein is more sensitive to abi3-7 mutation than AtEm1. However both mRNAs are considerably reduced in this mutant. Their distribution is also differentially affected. These results provide genetic evidence for the importance of the conserved B2 domain for ABI3 function in vivo.

The BANYULS gene encodes a DFR-like protein and is a marker of early seed coat development.

Mutations in the BANYULS (BAN) gene lead to precocious accumulation of anthocyanins in immature seed coat in Arabidopsis. The ban -1 allele has been isolated from a collection of T-DNA transformants and found to be tagged by the integrative molecule. The sequencing of wild-type and two independent mutant alleles confirmed the identity of the gene. Analysis of the full-length cDNA sequence revealed an open reading frame encoding a 342 amino acid protein which shared strong similarities with DFR and other enzymes of the phenylpropanoid biosynthesis pathway. BAN expression was restricted to the endothelium of immature seeds at the pre-globular to early globular stages of development as predicted from the maternal inheritance of the phenotype, and therefore represents a marker for early differentiation and development of the seed coat. BAN is probably involved in a metabolic channelling between the production of anthocyanins and pro-anthocyanidins in the seed coat.