Mapping QTLs for mineral accumulation and shoot dry biomass under different Zn nutritional conditions in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is one of the most important vegetables in China. Genetic dissection of leaf mineral accumulation and tolerance to Zn stress is important for the improvement of the nutritional quality of Chinese cabbage by breeding. A mapping population with 183 doubled haploid (DH) lines was used to study the genetics of mineral accumulation and the growth response to Zn. The genetic map was constructed based on 203 AFLPs, 58 SSRs, 22 SRAPs and four ESTPs. The concentration of 11 minerals was determined in leaves for 142 DH lines grown in an open field. In addition shoot dry biomass (SDB) under normal, deficient and excessive Zn nutritional conditions were investigated in hydroponics experiments. Ten QTLs, each explaining 11.1–17.1% of the Na, Mg, P, Al, Fe, Mn, Zn and Sr concentration variance, were identified by multiple-QTL model (MQM) mapping. One common QTL was found affecting SDB under normal, deficient and excessive Zn nutritional conditions. An additional QTL was detected for SDB under Zn excess stress only. These results offer insights into the genetic basis of leaf mineral accumulation and plant growth under Zn stress conditions in Chinese cabbage.     

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.     

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.     

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.     

Characterization of natural variation for zinc, iron and manganese accumulation and zinc exposure response in Brassica rapa L.

Brassica rapa L. is an important vegetable crop in eastern Asia. The objective of this study was to investigate the genetic variation in leaf Zn, Fe and Mn accumulation, Zn toxicity tolerance and Zn efficiency in B. rapa. In total 188 accessions were screened for their Zn-related characteristics in hydroponic culture. In experiment 1, mineral assays on 111 accessions grown under sufficient Zn supply (2 μM ZnSO₄) revealed a variation range of 23.2–155.9 μg g⁻¹ dry weight (d. wt.) for Zn, 60.3–350.1 μg g⁻¹ d. wt. for Fe and 20.9–53.3 μg g⁻¹ d. wt. for the Mn concentration in shoot. The investigation of tolerance to excessive Zn (800 μM ZnSO₄) on 158 accessions, by using visual toxicity symptom parameters (TSPs), identified different levels of tolerance in B. rapa. In experiment 2, a selected sub-set of accessions from experiment 1 was characterized in more detail for their mineral accumulation and tolerance to excessive Zn supply (100 μM and 300 μM ZnSO₄). In this experiment Zn tolerance (ZT) determined by relative root or shoot dry biomass varied about 2-fold. The same six accessions were also examined for Zn efficiency, determined as relative growth under 0 μM ZnSO₄ compared to 2 μM ZnSO₄. Zn efficiency varied 1.8-fold based on shoot dry biomass and 2.6-fold variation based on root dry biomass. Zn accumulation was strongly correlated with Mn and Fe accumulation both under sufficient and deficient Zn supply. In conclusion, there is substantial variation for Zn accumulation, Zn toxicity tolerance and Zn efficiency in Brassica rapa L., which would allow selective breeding for these traits.     

phenoVein—A Tool for Leaf Vein Segmentation and Analysis

Precise measurements of leaf vein traits are an important aspect of plant phenotyping for ecological and genetic research. Here, we present a powerful and user-friendly image analysis tool named phenoVein. It is dedicated to automated segmenting and analyzing of leaf veins in images acquired with different imaging modalities (microscope, macrophotography, etc.), including options for comfortable manual correction. Advanced image filtering emphasizes veins from the background and compensates for local brightness inhomogeneities. The most important traits being calculated are total vein length, vein density, piecewise vein lengths and widths, areole area, and skeleton graph statistics, like the number of branching or ending points. For the determination of vein widths, a model-based vein edge estimation approach has been implemented. Validation was performed for the measurement of vein length, vein width, and vein density of Arabidopsis (Arabidopsis thaliana), proving the reliability of phenoVein. We demonstrate the power of phenoVein on a set of previously described vein structure mutants of Arabidopsis (hemivenata, ondulata3, and asymmetric leaves2-101) compared with wild-type accessions Columbia-0 and Landsberg erecta-0. phenoVein is freely available as open-source software.Leaf veins are an important aspect of leaf structure and responsible for both the mechanical support of leaves and the long-distance transport of water, nutrients, and photoassimilates (Onoda et al., 2011; Malinowski, 2013). The molecular mechanisms by which vascular tissues acquire their identities are yet largely unknown (Roschzttardtz et al., 2014), and there is high interest in analyzing and evaluating traits of veins or leaf venation networks and their genetic regulation. The impact of vein density on photosynthesis is a major investigated topic (Sack and Scoffoni, 2013). During the last decade, a positive correlation between leaf venation and photosynthesis has been observed (Sack and Holbrook, 2006; Brodribb et al., 2007). An optimization of photosynthetic rates was shown to occur by spatial coordination between leaf vein and stomatal densities (Zhang et al., 2012; Carins Murphy et al., 2014; Fiorin et al., 2015). Additionally, there is interest in the impact of vein density on interveinal distances (Dengler et al., 1994; McKown and Dengler, 2009) and the effect of climate, habitat, or growth form on vein density (Sack and Scoffoni, 2013; Scoffoni et al., 2015) or vein width with respect to leaf hydraulic conductance (Feild and Brodribb, 2013; Xiong et al., 2015). Other researchers are particularly interested in the evolution from C3 to C4 plants, which requires higher vein density (Gowik and Westhoff, 2011) and led to selecting for variation of vein density within species (e.g. in a mutant collection by Feldman et al., 2014).Leaf venation studies analyzing traits of veins and venation networks are generally performed on microscopic images of leaves that are properly cleared after harvest. For very small leaves, e.g. the cotyledons or the first leaves (leaves 2–5) of Arabidopsis (Arabidopsis thaliana), basic traits, such as total vein length or vein density (vein length per leaf area), can be achieved manually. However, for larger leaves, manual vein segmentation may become tedious, and at least partially automated analysis is needed for studies on large series of leaf collections. Furthermore, the quantification of vein widths and in particular mean values of vein width of certain vein pieces of interest can hardly be achieved manually. Dedicated image processing tools are, therefore, needed to support researchers for fast and reliable data analysis.A number of software tools have been published that are either specifically made or adapted to analyze leaf veins. These programs have some common properties, like image processing functionalities for vein/areole segmentation and trait extraction. However, they differ in handling strategies or vein parameter analysis methods. A general overview on plant image analysis tools is collected in an online database at http://www.plant-image-analysis.org (Lobet et al., 2013). Programs allowing automated or semiautomated analysis of leaf venation parameters are, for example, a method to extract leaf venation patterns (Rolland-Lagan et al., 2009), the leaf extraction and analysis framework graphical user interface LeafGUI (Price et al., 2011), the leaf image analysis interface LIMANI (Dhondt et al., 2012), the user-interactive vessel generation analysis tool VESGEN (Vickerman et al., 2009; Parsons-Wingerter et al., 2014), and the software network extraction from images NEFI (Dirnberger et al., 2015). Nevertheless, for the analysis of large-scale leaf vein phenotyping experiments, there are certain needs that are only partly covered by each of the approaches and programs mentioned above. Specifically, the following properties are needed: (1) automated vein segmentation with optional manual correction; (2) invariance of the segmentation procedure to inhomogeneous illumination or brightness variations in the leaf image; (3) automated determination of total vein length and projected leaf area; (4) a well-defined and automated determination of vein widths, which is, as far as possible, independent of user chosen thresholds; (5) ability to process large high-resolution images of whole leafs; and (6) full transparency of the source code as well as offline availability of the tool. To provide these functionalities, we developed the user-friendly analysis tool phenoVein. It features automated leaf vein segmentation based on advanced image filtering techniques and includes determination of various vein traits, particularly a model-based vein width estimation. phenoVein allows easy and fast visual control and manual correction on the automatically achieved skeleton of the veins enabled by a real-time overlay of the segmented leaf vein structures on the original image. The length measurement algorithm of phenoVein was validated against complete manual segmentation. We evaluated the impact of image resolution on the results, which has recently been discussed (Price et al., 2014; Sack et al., 2014), and tested whether the orientation (angle) of a leaf on an image may affect the results as suspected from image analysis theory on binary skeleton length measurements (Russ, 2011). To show the powerful phenotyping capabilities of phenoVein, we analyzed the venation traits of leaves of Arabidopsis at different developmental stages (cotyledons, pooled leaves 1 + 2, and leaf 6) harvested from previously described venation mutants and corresponding wild-type lines: asymmetric leaves2-101 (as2-101), ondulata3 (ond3), and hemivenata2 (hve-2) versus Columbia-0 (Col-0) and Landsberg erecta-0 (Ler-0; Semiarti et al., 2001; Alonso-Peral et al., 2006; Robles et al., 2010; Pérez-Pérez et al., 2011). We offer the source code of phenoVein to the public as open-source software that can be further adapted or improved (for details, see “Materials and Methods”).     

The absence of histone H2B monoubiquitination in the Arabidopsis hub1 (rdo4) mutant reveals a role for chromatin remodeling in seed dormancy

Seed dormancy is defined as the failure of a viable seed to germinate under favorable conditions. Besides playing an adaptive role in nature by optimizing germination to the most suitable time, a tight control of dormancy is important in crop plants. Extensive genetic and physiological studies have identified the involvement of several factors, but the molecular mechanisms underlying this process are still largely unknown. We cloned the HISTONE MONOUBIQUITINATION1 (HUB1) gene, of which the mutant (previously identified as reduced dormancy4) has reduced seed dormancy and several pleiotropic phenotypes. HUB1 encodes a C3HC4 RING finger protein. The Arabidopsis thaliana genome contains one HUB1 homolog, which we named HUB2. The hub2 mutant also has reduced seed dormancy and is not redundant with hub1. Homologs of HUB1 and HUB2 in other species are required for histone H2B monoubiquitination. In agreement with this, the ubiquitinated form of histone H2B could not be detected in the hub1 and hub2 mutants. In yeast and human cells, histone H2B monoubiquitination is associated with actively transcribed genes. The hub1 mutant showed altered expression levels for several dormancy-related genes. We propose a role for chromatin remodeling in seed dormancy by H2B monoubiquitination through HUB1 and HUB2.     

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.