Functional evidence on the involvement of the MADS-box gene MdDAM4 in bud dormancy regulation in apple.

Over the course of the year, temperate trees experience extremes in temperature and day length. In order to protect themselves from frost damage in winter, they enter a dormant state with no visible growth where all leaves are shed and buds are dormant. Also the young floral tissues need to withstand harsh winter conditions, as temperature fruit trees like apple develop their flower buds in the previous year of fruit development. So far, the genetic control of induction and release of dormancy is not fully understood. However, the transcription factor family of DORMANCY-Associated MADS-box (DAM) genes plays a major role in the control of winter dormancy. One of these genes is MdDAM4. This gene is expressed in the early phase of bud dormancy, but little is known about its function. Six transgenic apple lines were produced to study the function of MdDAM4 in apple. For plant transformation, the binary plasmid vector p9oN-35s-MdDAM4 was used that contains the coding sequence of MdDAM4 driven by the 35S promoter. Transgenicity of the lines was proven by PCR and southern hybridization. Based on siRNA sequencing and phenotypic observations, it was concluded that line M2024 overexpresses MdDAM4 whereas the gene is silenced in all other lines. Phenotyping of the transgenic lines provided evidence that the overexpression of MdDAM4 leads to an earlier induction and a later release of dormancy. Silencing this gene had exactly the opposite effects and thereby led to an increased duration of the vegetation period. Expression experiments revealed genes that were either potentially repressed or activated by MdDAM4. Among the potentially suppressed genes were several homologs of the cytokinin oxidase 5 (CKX5), five LOX homologs, and several expansins, which may indicate a link between MdDAM4 and the control of leaf senescence. Among the potentially activated genes is MdDAM1, which is in line with observed expression patterns during winter dormancy. MdDAM2, which shows little expression during endodormancy also appears to be activated by MdDAM4. Overall, this study provides experimental evidence with transgenic apple trees for MdDAM4 being an important regulator of the onset of bud dormancy in apple.

LTP2 hypomorphs show genotype-by-environment interaction in early seedling traits in Arabidopsis thaliana.

Isogenic individuals can display seemingly stochastic phenotypic differences, limiting the accuracy of genotype-to-phenotype predictions. The extent of this phenotypic variation depends in part on genetic background, raising questions about the genes involved in controlling stochastic phenotypic variation. Focusing on early seedling traits in Arabidopsis thaliana, we found that hypomorphs of the cuticle-related gene LIPID TRANSFER PROTEIN 2 (LTP2) greatly increased variation in seedling phenotypes, including hypocotyl length, gravitropism and cuticle permeability. Many ltp2 hypocotyls were significantly shorter than wild-type hypocotyls while others resembled the wild-type. Differences in epidermal properties and gene expression between ltp2 seedlings with long and short hypocotyls suggest a loss of cuticle integrity as the primary determinant of the observed phenotypic variation. We identified environmental conditions that reveal or mask the increased variation in ltp2 hypomorphs and found that increased expression of its closest paralog LTP1 is necessary for ltp2 phenotypes. Our results illustrate how decreased expression of a single gene can generate starkly increased phenotypic variation in isogenic individuals in response to an environmental challenge.

The structure of the tetraploid sour cherry 'Schattenmorelle' (Prunus cerasus L.) genome reveals insights into its segmental allopolyploid nature.

Sour cherry (Prunus cerasus L.) is an important allotetraploid cherry species that evolved in the Caspian Sea and Black Sea regions from a hybridization of the tetraploid ground cherry (Prunus fruticosa Pall.) and an unreduced pollen of the diploid sweet cherry (P. avium L.) ancestor. Details of when and where the evolution of this species occurred are unclear, as well as the effect of hybridization on the genome structure. To gain insight, the genome of the sour cherry cultivar 'Schattenmorelle' was sequenced using Illumina NovaSeqTM and Oxford Nanopore long-read technologies, resulting in a ~629-Mbp pseudomolecule reference genome. The genome could be separated into two subgenomes, with subgenome PceS_a originating from P. avium and subgenome PceS_f originating from P. fruticosa. The genome also showed size reduction compared to ancestral species and traces of homoeologous sequence exchanges throughout. Comparative analysis confirmed that the genome of sour cherry is segmental allotetraploid and evolved very recently in the past.

Pan-European study of genotypes and phenotypes in the Arabidopsis relative Cardamine hirsuta reveals how adaptation, demography, and development shape diversity patterns.

We study natural DNA polymorphisms and associated phenotypes in the Arabidopsis relative Cardamine hirsuta. We observed strong genetic differentiation among several ancestry groups and broader distribution of Iberian relict strains in European C. hirsuta compared to Arabidopsis. We found synchronization between vegetative and reproductive development and a pervasive role for heterochronic pathways in shaping C. hirsuta natural variation. A single, fast-cycling ChFRIGIDA allele evolved adaptively allowing range expansion from glacial refugia, unlike Arabidopsis where multiple FRIGIDA haplotypes were involved. The Azores islands, where Arabidopsis is scarce, are a hotspot for C. hirsuta diversity. We identified a quantitative trait locus (QTL) in the heterochronic SPL9 transcription factor as a determinant of an Azorean morphotype. This QTL shows evidence for positive selection, and its distribution mirrors a climate gradient that broadly shaped the Azorean flora. Overall, we establish a framework to explore how the interplay of adaptation, demography, and development shaped diversity patterns of 2 related plant species.

LTP2 hypomorphs show genotype-by-environment interaction in early seedling traits in Arabidopsis thaliana.

Isogenic individuals can display seemingly stochastic phenotypic differences, limiting the accuracy of genotype-to-phenotype predictions. The extent of this phenotypic variation depends in part on genetic background, raising questions about the genes involved in controlling stochastic phenotypic variation. Focusing on early seedling traits in Arabidopsis thaliana, we found that hypomorphs of the cuticle-related gene LTP2 greatly increased variation in seedling phenotypes, including hypocotyl length, gravitropism and cuticle permeability. Many ltp2 hypocotyls were significantly shorter than wild-type hypocotyls while others resembled the wild type. Differences in epidermal properties and gene expression between ltp2 seedlings with long and short hypocotyls suggest a loss of cuticle integrity as the primary determinant of the observed phenotypic variation. We identified environmental conditions that reveal or mask the increased variation in ltp2 hypomorphs, and found that increased expression of its closest paralog LTP1 is necessary for ltp2 phenotypes. Our results illustrate how decreased expression of a single gene can generate starkly increased phenotypic variation in isogenic individuals in response to an environmental challenge.

Exploring epigenetic variation for breeding climate resilient apple crops.

Climate change with warmer winter and spring temperatures poses major challenges to apple fruit production. Long-term observations confirm the trend toward earlier flowering, which leads to an increased risk of frost damage. New breeding strategies are needed to generate cultivars that are able to stay largely unaffected by warmer temperatures. Recently, epigenetic variation has been proposed as a new resource for breeding purposes and seems suitable in principle for apple breeding. However, to serve as a new resource for apple breeding, it is necessary to clarify whether epigenetic variation can be induced by the environment, whether it can create phenotypic variation, and whether this variation is stable across generations. In this brief review, we summarize the impact of climate change on the timing of apple phenology, highlight how epigenetic variation can potentially support novel breeding strategies, and point out important features of epigenetic variation that are required for its application in breeding programs.

Recent Developments and Strategies for the Application of Agrobacterium-Mediated Transformation of Apple Malus × domestica Borkh.

Genetic transformation has become an important tool in plant genome research over the last three decades. This applies not only to model plants such as Arabidopsis thaliana but also increasingly to cultivated plants, where the establishment of transformation methods could still pose many problems. One of such plants is the apple (Malus spp.), the most important fruit of the temperate climate zone. Although the genetic transformation of apple using Agrobacterium tumefaciens has been possible since 1989, only a few research groups worldwide have successfully applied this technology, and efficiency remains poor. Nevertheless, there have been some developments, especially in recent years, which allowed for the expansion of the toolbox of breeders and breeding researchers. This review article attempts to summarize recent developments in the Agrobacterium-mediated transformation strategies of apple. In addition to the use of different tissues and media for transformation, agroinfiltration, as well as pre-transformation with a Baby boom transcription factor are notable successes that have improved transformation efficiency in apple. Further, we highlight targeted gene silencing applications. Besides the classical strategies of RNAi-based silencing by stable transformation with hairpin gene constructs, optimized protocols for virus-induced gene silencing (VIGS) and artificial micro RNAs (amiRNAs) have emerged as powerful technologies for silencing genes of interest. Success has also been achieved in establishing methods for targeted genome editing (GE). For example, it was recently possible for the first time to generate a homohistont GE line into which a biallelic mutation was specifically inserted in a target gene. In addition to these methods, which are primarily aimed at increasing transformation efficiency, improving the precision of genetic modification and reducing the time required, methods are also discussed in which genetically modified plants are used for breeding purposes. In particular, the current state of the rapid crop cycle breeding system and its applications will be presented.

Profiling of phytohormones in apple fruit and buds regarding their role as potential regulators of flower bud formation.

Apple (Malus × domestica Borkh.) cropping behavior, if not regulated, is often manifested by high yields of small-sized fruit in so called ON-years, which are usually followed by strongly reduced crop loads in OFF-years. Such cropping pattern is defined as biennial bearing and causes significant losses in apple production. The growth of apple fruit overlaps with the formation of flower buds, which remain dormant until the following spring. Earlier works proposed that some fruit-derived mobile compounds, as e.g., phytohormones, could suppress flower bud formation that thereby leads to biennial bearing. We addressed this hypothesis by analyzing 39 phytohormones in apple seeds, fruit flesh and by measuring phytohormone export from the fruits of the biennial bearing cultivar 'Fuji' and of the regular bearing cultivar 'Gala'. Moreover, we analyzed the same compounds in bourse buds from fruiting (ON-trees) and non-fruiting (OFF-trees) spurs of both apple cultivars over the period of flower bud formation. Our results showed that apple fruit exported at least 14 phytohormones including indole-3-acetic acid and gibberellin A3; however, their influence on flower bud formation was inconclusive. A gibberellin-like compound, which was detected exclusively in bourse buds, was significantly more abundant in bourse buds from ON-trees compared with OFF-trees. Cultivar differences were marked by the accumulation of trans-zeatin-O-glucoside in bourse buds of 'Gala' ON-trees, whereas the levels of this compound in 'Gala' OFF were significantly lower and comparable to those in 'Fuji' ON- and OFF-trees. Particular phytohormones including five cytokinin forms as well as abscisic acid and its degradation products had higher levels in bourse buds from OFF-trees compared with ON-trees and were therefore proposed as potential promotors of flower bud initiation. The work discusses regulatory roles of phytohormones in flower bud formation in apple based on the novel and to date most comprehensive phytohormone profiles of apple fruit and buds.

The draft chromosome-level genome assembly of tetraploid ground cherry (Prunus fruticosa Pall.) from long reads.

Cherries are stone fruits and belong to the economically important plant family of Rosaceae with worldwide cultivation of different species. The ground cherry, Prunus fruticosa Pall., is an ancestor of cultivated sour cherry, an important tetraploid cherry species. Here, we present a long read chromosome-level draft genome assembly and related plastid sequences using the Oxford Nanopore Technology PromethION platform and R10.3 pore type. We generated a final consensus genome sequence of 366 Mb comprising eight chromosomes. The N50 scaffold was ~44 Mb with the longest chromosome being 66.5 Mb. The chloroplast and mitochondrial genomes were 158,217 bp and 383,281 bp long, which is in accordance with previously published plastid sequences. This is the first report of the genome of ground cherry (P. fruticosa) sequenced by long read technology only. The datasets obtained from this study provide a foundation for future breeding, molecular and evolutionary analysis in Prunus studies.

Time-Resolved Analysis of Candidate Gene Expression and Ambient Temperature During Bud Dormancy in Apple.

Winter dormancy - a period of low metabolic activity and no visible growth - appears as an adaptation to harsh winter conditions and can be divided into different phases. It is tightly controlled by environmental cues, with ambient temperature playing a major role. During endodormancy, a cultivar-specific amount of cold needs to be perceived, and during ecodormancy, heat hours accumulate before bud burst and anthesis in spring. Expression analysis, performed in several key fruit tree species, proved to be very useful in elucidating the molecular control of onset and release of dormancy. However, the time resolution of these experiments has been limited. Therefore, in this study, dense time-series expression analysis was conducted for 40 candidate genes involved in dormancy control, under the cool-temperate climate conditions in Dresden. Samples were taken from the cultivars 'Pinova' and 'Gala,' which differ in flowering time. The set of candidate genes included well-established dormancy genes such as DAM genes, MdFLC-like, MdICE1, MdPRE 1, and MdPIF4. Furthermore, we tested genes from dormancy-associated pathways including the brassinosteroid, gibberellic acid, abscisic acid (ABA), cytokinin response, and respiratory stress pathways. The expression patterns of well-established dormancy genes were confirmed and could be associated with specific dormancy phases. In addition, less well-known transcription factors and genes of the ABA signaling pathway showed associations with dormancy progression. The three ABA signaling genes HAB1_chr15, HAI3, and ABF2 showed a local minimum of gene expression in proximity of the endodormancy to ecodormancy transition. The number of sampling points allowed us to correlate expression values with temperature data, which revealed significant correlations of ambient temperature with the expression of the Malus domestica genes MdICE1, MdPIF4, MdFLC-like, HAB1chr15, and the type-B cytokinin response regulator BRR9. Interestingly, the slope of the linear correlation of temperature with the expression of MdPIF4 differed between cultivars. Whether the strength of inducibility of MdPIF4 expression by low temperature differs between the 'Pinova' and 'Gala' alleles needs to be tested further.

Phyllotaxis: is the golden angle optimal for light capture?

Phyllotactic patterns are some of the most conspicuous in nature. To create these patterns plants must control the divergence angle between the appearance of successive organs, sometimes to within a fraction of a degree. The most common angle is the Fibonacci or golden angle, and its prevalence has led to the hypothesis that it has been selected by evolution as optimal for plants with respect to some fitness benefits, such as light capture. We explore arguments for and against this idea with computer models. We have used both idealized and scanned leaves from Arabidopsis thaliana and Cardamine hirsuta to measure the overlapping leaf area of simulated plants after varying parameters such as leaf shape, incident light angles, and other leaf traits. We find that other angles generated by Fibonacci-like series found in nature are equally optimal for light capture, and therefore should be under similar evolutionary pressure. Our findings suggest that the iterative mechanism for organ positioning itself is a more likely target for evolutionary pressure, rather than a specific divergence angle, and our model demonstrates that the heteroblastic progression of leaf shape in A. thaliana can provide a potential fitness benefit via light capture.

MowJoe: a method for automated-high throughput dissected leaf phenotyping.

BACKGROUND: Accurate and automated phenotyping of leaf images is necessary for high throughput studies of leaf form like genome-wide association analysis and other forms of quantitative trait locus mapping. Dissected leaves (also referred to as compound) that are subdivided into individual units are an attractive system to study diversification of form. However, there are only few software tools for their automated analysis. Thus, high-throughput image processing algorithms are needed that can partition these leaves in their phenotypically relevant units and calculate morphological features based on these units. RESULTS: We have developed MowJoe, an image processing algorithm that dissects a dissected leaf into leaflets, petiolule, rachis and petioles. It employs image skeletonization to convert leaves into graphs, and thereafter applies algorithms operating on graph structures. This partitioning of a leaf allows the derivation of morphological features such as leaf size, or eccentricity of leaflets. Furthermore, MowJoe automatically places landmarks onto the terminal leaflet that can be used for further leaf shape analysis. It generates specific output files that can directly be imported into downstream shape analysis tools. We applied the algorithm to two accessions of Cardamine hirsuta and show that our features are able to robustly discriminate between these accessions. CONCLUSION: MowJoe is a tool for the semi-automated, quantitative high throughput shape analysis of dissected leaf images. It provides the statistical power for the detection of the genetic basis of quantitative morphological variations.

Genomic Rearrangements in Arabidopsis Considered as Quantitative Traits.

To understand the population genetics of structural variants and their effects on phenotypes, we developed an approach to mapping structural variants that segregate in a population sequenced at low coverage. We avoid calling structural variants directly. Instead, the evidence for a potential structural variant at a locus is indicated by variation in the counts of short-reads that map anomalously to that locus. These structural variant traits are treated as quantitative traits and mapped genetically, analogously to a gene expression study. Association between a structural variant trait at one locus, and genotypes at a distant locus indicate the origin and target of a transposition. Using ultra-low-coverage (0.3×) population sequence data from 488 recombinant inbred Arabidopsis thaliana genomes, we identified 6502 segregating structural variants. Remarkably, 25% of these were transpositions. While many structural variants cannot be delineated precisely, we validated 83% of 44 predicted transposition breakpoints by polymerase chain reaction. We show that specific structural variants may be causative for quantitative trait loci for germination and resistance to infection by the fungus Albugo laibachii, isolate Nc14. Further we show that the phenotypic heritability attributable to read-mapping anomalies differs from, and, in the case of time to germination and bolting, exceeds that due to standard genetic variation. Genes within structural variants are also more likely to be silenced or dysregulated. This approach complements the prevalent strategy of structural variant discovery in fewer individuals sequenced at high coverage. It is generally applicable to large populations sequenced at low-coverage, and is particularly suited to mapping transpositions.

Heterochrony underpins natural variation in Cardamine hirsuta leaf form.

A key problem in biology is whether the same processes underlie morphological variation between and within species. Here, by using plant leaves as an example, we show that the causes of diversity at these two evolutionary scales can be divergent. Some species like the model plant Arabidopsis thaliana have simple leaves, whereas others like the A. thaliana relative Cardamine hirsuta bear complex leaves comprising leaflets. Previous work has shown that these interspecific differences result mostly from variation in local tissue growth and patterning. Now, by cloning and characterizing a quantitative trait locus (QTL) for C. hirsuta leaf shape, we find that a different process, age-dependent progression of leaf form, underlies variation in this trait within species. This QTL effect is caused by cis-regulatory variation in the floral repressor ChFLC, such that genotypes with low-expressing ChFLC alleles show both early flowering and accelerated age-dependent changes in leaf form, including faster leaflet production. We provide evidence that this mechanism coordinates leaf development with reproductive timing and may help to optimize resource allocation to the next generation.

Mapping and dynamics of regulatory DNA and transcription factor networks in A. thaliana.

Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis-regulatory DNA and its dynamics. We mapped DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and used genomic footprinting to delineate ∼ 700,000 sites of in vivo transcription factor (TF) occupancy at nucleotide resolution. We show that variation associated with 72 diverse quantitative phenotypes localizes within DHSs. TF footprints encode an extensive cis-regulatory lexicon subject to recent evolutionary pressures, and widespread TF binding within exons may have shaped codon usage patterns. The architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. We analyzed regulatory landscape dynamics during heat shock and photomorphogenesis, disclosing thousands of environmentally sensitive elements and enabling mapping of key TF regulatory circuits underlying these fundamental responses. Our results provide an extensive resource for the study of A. thaliana gene regulation and functional biology.

Molecular mechanisms of robustness in plants.

Robustness, the ability of organisms to buffer phenotypes against perturbations, has drawn renewed interest among developmental biologists and geneticists. A growing body of research supports an important role of robustness in the genotype to phenotype translation, with far-reaching implications for evolutionary processes and disease susceptibility. Similar to animals and fungi, plant robustness is a function of genetic network architecture. Most perturbations are buffered; however, perturbation of network hubs destabilizes many traits. Here, we review recent advances in identifying molecular robustness mechanisms in plants that have been enabled by a combination of classical genetics and population genetics with genome-scale data.

Cis-regulatory changes at FLOWERING LOCUS T mediate natural variation in flowering responses of Arabidopsis thaliana.

Flowering time, a critical adaptive trait, is modulated by several environmental cues. These external signals converge on a small set of genes that in turn mediate the flowering response. Mutant analysis and subsequent molecular studies have revealed that one of these integrator genes, FLOWERING LOCUS T (FT), responds to photoperiod and temperature cues, two environmental parameters that greatly influence flowering time. As the central player in the transition to flowering, the protein coding sequence of FT and its function are highly conserved across species. Using QTL mapping with a new advanced intercross-recombinant inbred line (AI-RIL) population, we show that a QTL tightly linked to FT contributes to natural variation in the flowering response to the combined effects of photoperiod and ambient temperature. Using heterogeneous inbred families (HIF) and introgression lines, we fine map the QTL to a 6.7 kb fragment in the FT promoter. We confirm by quantitative complementation that FT has differential activity in the two parental strains. Further support for FT underlying the QTL comes from a new approach, quantitative knockdown with artificial microRNAs (amiRNAs). Consistent with the causal sequence polymorphism being in the promoter, we find that the QTL affects FT expression. Taken together, these results indicate that allelic variation at pathway integrator genes such as FT can underlie phenotypic variability and that this may be achieved through cis-regulatory changes.

Autoimmune response as a mechanism for a Dobzhansky-Muller-type incompatibility syndrome in plants.

Epistatic interactions between genes are a major factor in evolution. Hybrid necrosis is an example of a deleterious phenotype caused by epistatic interactions that is observed in many intra- and interspecific plant hybrids. A large number of hybrid necrosis cases share phenotypic similarities, suggesting a common underlying mechanism across a wide range of plant species. Here, we report that approximately 2% of intraspecific crosses in Arabidopsis thaliana yield F1 progeny that express necrosis when grown under conditions typical of their natural habitats. We show that several independent cases result from epistatic interactions that trigger autoimmune-like responses. In at least one case, an allele of an NB-LRR disease resistance gene homolog is both necessary and sufficient for the induction of hybrid necrosis, when combined with a specific allele at a second locus. The A. thaliana cases provide insights into the molecular causes of hybrid necrosis, and serve as a model for further investigation of intra- and interspecific incompatibilities caused by a simple epistatic interaction. Moreover, our finding that plant immune-system genes are involved in hybrid necrosis suggests that selective pressures related to host-pathogen conflict might cause the evolution of gene flow barriers in plants.

Potent induction of Arabidopsis thaliana flowering by elevated growth temperature.

The transition to flowering is an important event in the plant life cycle and is modulated by several environmental factors including photoperiod, light quality, vernalization, and growth temperature, as well as biotic and abiotic stresses. In contrast to light and vernalization, little is known about the pathways that mediate the responses to other environmental variables. A mild increase in growth temperature, from 23 degrees C to 27 degrees C, is equally efficient in inducing flowering of Arabidopsis plants grown in 8-h short days as is transfer to 16-h long days. There is extensive natural variation in this response, and we identify strains with contrasting thermal reaction norms. Exploiting this natural variation, we show that FLOWERING LOCUS C potently suppresses thermal induction, and that the closely related floral repressor FLOWERING LOCUS M is a major-effect quantitative trait locus modulating thermosensitivity. Thermal induction does not require the photoperiod effector CONSTANS, acts upstream of the floral integrator FLOWERING LOCUS T, and depends on the hormone gibberellin. Analysis of mutants defective in salicylic acid biosynthesis suggests that thermal induction is independent of previously identified stress-signaling pathways. Microarray analyses confirm that the genomic responses to floral induction by photoperiod and temperature differ. Furthermore, we report that gene products that participate in RNA splicing are specifically affected by thermal induction. Above a critical threshold, even small changes in temperature can act as cues for the induction of flowering. This response has a genetic basis that is distinct from the known genetic pathways of floral transition, and appears to correlate with changes in RNA processing.

Diversity of flowering responses in wild Arabidopsis thaliana strains.

Although multiple environmental cues regulate the transition to flowering in Arabidopsis thaliana, previous studies have suggested that wild A. thaliana accessions fall primarily into two classes, distinguished by their requirement for vernalization (extended winter-like temperatures), which enables rapid flowering under long days. Much of the difference in vernalization response is apparently due to variation at two epistatically acting loci, FRI and FLC. We present the response of over 150 wild accessions to three different environmental variables. In long days, FLC is among those genes whose expression is most highly correlated with flowering. In short days, FRI and FLC are less important, although their contribution is still significant. In addition, there is considerable variation not only in vernalization response, but also in the response to differences in day length or ambient growth temperature. The identification of accessions that flower relatively early or late in specific environments suggests that many of the flowering-time pathways identified by mutagenesis, such as those that respond to day length, contribute to flowering-time variation in the wild. In contrast to differences in vernalization requirement, which are mainly mediated by FRI and FLC, it seems that variation in these other pathways is due to allelic effects at several different loci.