Chris Dardick

Plum (Prunus domestica) trees transformed with poplar FT1 result in altered architecture, dormancy requirement, and continuous flowering.

The Flowering Locus T1 (FT1) gene from Populus trichocarpa under the control of the 35S promoter was transformed into European plum (Prunus domestica L). Transgenic plants expressing higher levels of FT flowered and produced fruits in the greenhouse within 1 to 10 months. FT plums did not enter dormancy after cold or short day treatments yet field planted FT plums remained winter hardy down to at least −10°C. The plants also displayed pleiotropic phenotypes atypical for plum including shrub-type growth habit and panicle flower architecture. The flowering and fruiting phenotype was found to be continuous in the greenhouse but limited to spring and fall in the field. The pattern of flowering in the field correlated with lower daily temperatures. This apparent temperature effect was subsequently confirmed in growth chamber studies. The pleitropic phenotypes associated with FT1 expression in plum suggests a fundamental role of this gene in plant growth and development. This study demonstrates the potential for a single transgene event to markedly affect the vegetative and reproductive growth and development of an economically important temperate woody perennial crop. We suggest that FT1 may be a useful tool to modify temperate plants to changing climates and/or to adapt these crops to new growing areas.

Unique expression, processing regulation, and regulatory network of peach (Prunus persica) miRNAs

BackgroundMicroRNAs (miRNAs) have recently emerged as important gene regulators in plants. MiRNAs and their targets have been extensively studied in Arabidopsis and rice. However, relatively little is known about the characterization of miRNAs and their target genes in peach (Prunus persica), which is a complex crop with unique developmental programs.ResultsWe performed small RNA deep sequencing and identified 47 peach-specific and 47 known miRNAs or families with distinct expression patterns. Together, the identified miRNAs targeted 80 genes, many of which have not been reported previously. Like the model plant systems, peach has two of the three conserved trans-acting siRNA biogenesis pathways with similar mechanistic features and target specificity. Unique to peach, three of the miRNAs collectively target 49 MYBs, 19 of which are known to regulate phenylpropanoid metabolism, a key pathway associated with stone hardening and fruit color development, highlighting a critical role of miRNAs in the regulation of peach fruit development and ripening. We also found that the majority of the miRNAs were differentially regulated in different tissues, in part due to differential processing of miRNA precursors. Up to 16% of the peach-specific miRNAs were differentially processed from their precursors in a tissue specific fashion, which has been rarely observed in plant cells. The miRNA precursor processing activity appeared not to be coupled with its transcriptional activity but rather acted independently in peach.ConclusionsCollectively, the data characterizes the unique expression pattern and processing regulation of peach miRNAs and demonstrates the presence of a complex, multi-level miRNA regulatory network capable of targeting a wide variety of biological functions, including phenylpropanoid pathways which play a multifaceted spatial-temporal role in peach fruit development.

Transcriptomics of shading-induced and NAA-induced abscission in apple (Malus domestica) reveals a shared pathway involving reduced photosynthesis, alterations in carbohydrate transport and signaling and hormone crosstalk

BackgroundNaphthaleneacetic acid (NAA), a synthetic auxin analogue, is widely used as an effective thinner in apple orchards. When applied shortly after fruit set, some fruit abscise leading to improved fruit size and quality. However, the thinning results of NAA are inconsistent and difficult to predict, sometimes leading to excess fruit drop or insufficient thinning which are costly to growers. This unpredictability reflects our incomplete understanding of the mode of action of NAA in promoting fruit abscission.ResultsHere we compared NAA-induced fruit drop with that caused by shading via gene expression profiling performed on the fruit abscission zone (FAZ), sampled 1, 3, and 5 d after treatment. More than 700 genes with significant changes in transcript abundance were identified from NAA-treated FAZ. Combining results from both treatments, we found that genes associated with photosynthesis, cell cycle and membrane/cellular trafficking were downregulated. On the other hand, there was up-regulation of genes related to ABA, ethylene biosynthesis and signaling, cell wall degradation and programmed cell death. While the differentially expressed gene sets for NAA and shading treatments shared only 25% identity, NAA and shading showed substantial similarity with respect to the classes of genes identified. Specifically, photosynthesis, carbon utilization, ABA and ethylene pathways were affected in both NAA- and shading-induced young fruit abscission. Moreover, we found that NAA, similar to shading, directly interfered with leaf photosynthesis by repressing photosystem II (PSII) efficiency within 10 minutes of treatment, suggesting that NAA and shading induced some of the same early responses due to reduced photosynthesis, which concurred with changes in hormone signaling pathways and triggered fruit abscission.ConclusionsThis study provides an extensive transcriptome study and a good platform for further investigation of possible regulatory genes involved in the induction of young fruit abscission in apple, which will enable us to better understand the mechanism of fruit thinning and facilitate the selection of potential chemicals for the thinning programs in apple.

High transformation efficiency in plum ( Prunus domestica L.): a new tool for functional genomics studies in Prunus spp.

An improved Agrobacterium-mediated transformation protocol for plum (Prunus domestica L.) hypocotyl slices was developed based on the addition of 2,4-d to the co-cultivation medium. This method increased transformation efficiency up to 10xa0×xa0(42%) over previous reports with an average efficiency of 25% of hypocotyl slices producing transgenic plants. Timing of each step in the protocol was optimized producing self-rooted transgenic plants in the greenhouse in approximately 6xa0months. In order to test the system for its utility in functional genomic studies, we developed two hairpin constructs using a fragment of the peach (P. persica) Phytoene desaturase (PDS) gene. When A. tumefaciens with these constructs was used for targeted post-transcriptional gene silencing (PTGS), approximately 50% of the transformed plums were knockout PDS gene plants. The easy and efficient plum transformation protocol that we report here can be readily used for functional genomics studies in Prunus specifically, and Rosaceae and other woody species in general.

Non-arginine-aspartate (non-RD) kinases are associated with innate immune receptors that recognize conserved microbial signatures.

An important question in the field of plant-pathogen interactions is how the detection of pathogens is converted into an effective immune response. In recent years, substantial insight has been gained into the identities of both the plant receptors and the microbial molecules they recognize. Likewise, many of the downstream signaling proteins and transcriptions factors that activate defense responses have been characterized. However, the early molecular events that comprise recognition and how defense signaling specificity is achieved are not as well understood. In this review we discuss the significance of non-arginine-aspartate (non-RD) kinases, a subclass of kinases that are often found in association with pattern recognition receptors (PRRs).

PpeTAC1 promotes the horizontal growth of branches in peach trees and is a member of a functionally conserved gene family found in diverse plants species

Trees are capable of tremendous architectural plasticity, allowing them to maximize their light exposure under highly competitive environments. One key component of tree architecture is the branch angle, yet little is known about the molecular basis for the spatial patterning of branches in trees. Here, we report the identification of a candidate gene for the br mutation in Prunus persica (peach) associated with vertically oriented growth of branches, referred to as pillar or broomy. Ppa010082, annotated as hypothetical protein in the peach genome sequence, was identified as a candidate gene for br using a next generation sequence-based mapping approach. Sequence similarity searches identified rice TAC1 (tiller angle control 1) as a putative ortholog, and we thus named it PpeTAC1. In monocots, TAC1 is known to lead to less compact growth by increasing the tiller angle. In Arabidopsis, an attac1 mutant showed more vertical branch growth angles, suggesting that the gene functions universally to promote the horizontal growth of branches. TAC1 genes belong to a gene family (here named IGT for a shared conserved motif) found in all plant genomes, consisting of two clades: one containing TAC1-like genes; the other containing LAZY1, which contains an EAR motif, and promotes vertical shoot growth in Oryza sativa (rice) and Arabidopsis through influencing polar auxin transport. The data suggest that IGT genes are ancient, and play conserved roles in determining shoot growth angles in plants. Understanding how IGT genes modulate branch angles will provide insights into how different architectural growth habits evolved in terrestrial plants.

Genetic engineering of Plum pox virus resistance: ‘HoneySweet’ plum—from concept to product

Sharka disease, caused by Plum pox virus (PPV) was first recorded in Bulgaria during the early twentieth century and since that first report, the disease has progressively spread throughout Europe and more recently to Asia, Africa, North and South America. Few PPV resistance genes have been found to naturally occur in Prunus and this has led to the investigation of biotech approaches to the development of resistance through genetic engineering (GE). A notable example of the utility of this approach is ‘HoneySweet’ plum. PPV protection in this case is based on RNA interference (RNAi) and resistance has been shown to be highly effective, stable, durable, and heritable as a dominant trait. Extensive testing and risk assessment of ‘HoneySweet’ in laboratory, greenhouse and in the field for over 20xa0years has demonstrated not only the effectiveness but also the safety of the technology. ‘HoneySweet’ has been cleared for cultivation in the USA. By the appropriate regulatory agencies. The development and regulatory approval of ‘HoneySweet’ demonstrate the ability of RNAi technology to contribute to the sustainability of stone fruit production in regions impacted by PPV. Although it has taken almost 100xa0years since the identification of sharka, we are now able to effectively protect stone fruit species against this disease through the application of GE.

Genomic analysis reveals MATH gene(s) as candidate(s) for Plum pox virus (PPV) resistance in apricot (Prunus armeniaca L.)

Sharka disease, caused by Plum pox virus (PPV), is the most important viral disease affecting Prunus species. A major PPV resistance locus (PPVres) has been mapped to the upper part of apricot (Prunus armeniaca) linkage group 1. In this study, a physical map of the PPVres locus in the PPV-resistant cultivar Goldrich was constructed. Bacterial artificial chromosome (BAC) clones belonging to the resistant haplotype contig were sequenced using 454/GS-FLX Titanium technology. Concurrently, the whole genome of seven apricot varieties (three PPV-resistant and four PPV-susceptible) and two PPV-susceptible apricot relatives (P.u2009sibirica var. davidiana and P.u2009mume) were obtained using the Illumina-HiSeq2000 platform. Single nucleotide polymorphisms (SNPs) within the mapped interval, recorded from alignments against the peach genome, allowed us to narrow down the PPVres locus to a region of ∼196u2009kb. Searches for polymorphisms linked in coupling with the resistance led to the identification of 68 variants within 23 predicted transcripts according to peach genome annotation. Candidate resistance genes were ranked combining data from variant calling and predicted functions inferred from sequence homology. Together, the results suggest that members of a cluster of meprin and TRAF-C homology domain (MATHd)-containing proteins are the most likely candidate genes for PPV resistance in apricot. Interestingly, MATHd proteins are hypothesized to control long-distance movement (LDM) of potyviruses in Arabidopsis, and restriction for LDM is also a major component of PPV resistance in apricot. Although the PPV resistance gene(s) remains to be unambiguously identified, these results pave the way to the determination of the underlying mechanism and to the development of more accurate breeding strategies.

Evolution of the fruit endocarp: molecular mechanisms underlying adaptations in seed protection and dispersal strategies

Plant evolution is largely driven by adaptations in seed protection and dispersal strategies that allow diversification into new niches. This is evident by the tremendous variation in flowering and fruiting structures present both across and within different plant lineages. Within a single plant family a staggering variety of fruit types can be found such as fleshy fruits including berries, pomes, and drupes and dry fruit structures like achenes, capsules, and follicles. What are the evolutionary mechanisms that enable such dramatic shifts to occur in a relatively short period of time? This remains a fundamental question of plant biology today. On the surface it seems that these extreme differences in form and function must be the consequence of very different developmental programs that require unique sets of genes. Yet as we begin to decipher the molecular and genetic basis underlying fruit form it is becoming apparent that simple genetic changes in key developmental regulatory genes can have profound anatomical effects. In this review, we discuss recent advances in understanding the molecular mechanisms of fruit endocarp tissue differentiation that have contributed to species diversification within three plant lineages.

Molecular basis of angiosperm tree architecture

The architecture of trees greatly impacts the productivity of orchards and forestry plantations. Amassing greater knowledge on the molecular genetics that underlie tree form can benefit these industries, as well as contribute to basic knowledge of plant developmental biology. This review describes the fundamental components of branch architecture, a prominent aspect of tree structure, as well as genetic and hormonal influences inferred from studies in model plant systems and from trees with non-standard architectures. The bulk of the molecular and genetic data described here is from studies of fruit trees and poplar, as these species have been the primary subjects of investigation in this field of science.