Comparison of red raspberry and wild strawberry fruits reveals mechanisms of fruit type specification.

Belonging to Rosaceae, red raspberry (Rubus idaeus) and wild strawberry (Fragaria vesca) are closely related species with distinct fruit types. While the numerous ovaries become the juicy drupelet fruits in raspberry, their strawberry counterparts become dry and tasteless achenes. In contrast, while the strawberry receptacle, the stem tip, enlarges to become a red fruit, the raspberry receptacle shrinks and dries. The distinct fruit-forming ability of homologous organs in these 2 species allows us to investigate fruit type determination. We assembled and annotated the genome of red raspberry (R. idaeus) and characterized its fruit development morphologically and physiologically. Subsequently, transcriptomes of dissected and staged raspberry fruit tissues were compared to those of strawberry from a prior study. Class B MADS box gene expression was negatively associated with fruit-forming ability, which suggested a conserved inhibitory role of class B heterodimers, PISTILLATA/TM6 or PISTILLATA/APETALA3, for fruit formation. Additionally, the inability of strawberry ovaries to develop into fruit flesh was associated with highly expressed lignification genes and extensive lignification of the ovary pericarp. Finally, coexpressed gene clusters preferentially expressed in the dry strawberry achenes were enriched in "cell wall biosynthesis" and "ABA signaling," while coexpressed clusters preferentially expressed in the fleshy raspberry drupelets were enriched in "protein translation." Our work provides extensive genomic resources as well as several potential mechanisms underlying fruit type specification. These findings provide the framework for understanding the evolution of different fruit types, a defining feature of angiosperms.

Comparative transcriptomic analysis of apple and peach fruits: insights into fruit type specification.

Fruits represent key evolutionary innovations in angiosperms and exhibit diverse types adapted for seed dissemination. However, the mechanisms that underlie fruit type diversity are not understood. The Rosaceae family comprises many different fruit types, including 'pome' and 'drupe' fruits, and hence is an excellent family for investigating the genetic basis of fruit type specification. Using comparative transcriptomics, we investigated the molecular events that correlate with pome (apple) and drupe (peach) fleshy fruit development, focusing on the earliest stages of fruit initiation. We identified PI and TM6, MADS box genes whose expression negatively correlates with fruit flesh-forming tissues irrespective of fruit type. In addition, the MADS box gene FBP9 is expressed in fruit-forming tissues in both species, and was lost multiple times in the genomes of dry-fruit-forming eudicots including Arabidopsis. Network analysis reveals co-expression between FBP9 and photosynthesis genes in both apple and peach, suggesting that FBP9 and photosynthesis may both promote fleshy fruit development. The large transcriptomic datasets at the earliest stages of pome and drupe fruit development provide rich resources for comparative studies, and the work provides important insights into fruit-type specification.

Long-Term Efficacy and Safety of RNAi-Mediated Virus Resistance in 'HoneySweet' Plum.

Interfering RNA technology has been established as an effective strategy to protect plants against viral infection. Despite this success, interfering RNA (RNAi) has rarely been applied due to the regulatory barriers that confront genetically engineered plants and concerns over possible environmental and health risks posed by non-endogenous small RNAs. 'HoneySweet' was developed as a virus-resistant plum variety that is protected by an RNAi-mediated process against Sharka disease caused by the plum pox virus. 'HoneySweet' has been approved for cultivation in the United States but not in countries where the plum pox virus is endemic. In this study, we evaluated the long-term efficacy of virus resistance in 'HoneySweet,' the nature and stability of its sRNA profile, and the potential health risks of consuming 'HoneySweet' plums. Graft-challenged 'HoneySweet' trees carrying large non-transgenic infected limbs remained virus-free after more than 10 years in the field, and the viral sequences from the non-transgenic infected limbs showed no evidence of adaptation to the RNAi-based resistance. Small RNA profiling revealed that transgene-derived sRNA levels were stable across different environments and, on average, were more than 10 times lower than those present in symptom-less fruits from virus-infected trees. Comprehensive 90-day mouse feeding studies showed no adverse health impacts in mice, and there was no evidence for potential siRNA off-target pathologies predicted by comparisons of the most abundant transgene-derived sRNAs to the mouse genome. Collectively, the data confirmed that RNAi provides a highly effective, stable, and safe strategy to combat virus diseases in crop plants.

Robust Response to Plum pox virus Infection via Plant Biotechnology.

Our goal was to target silencing of the Plum pox virus coat protein (PPV CP) gene independently expressed in plants. Clone C-2 is a transgenic plum expressing CP. We introduced and verified, in planta, the effects of the inverse repeat of CP sequence split by a hairpin (IRSH) that was characterized in the HoneySweet plum. The IRSH construct was driven by two CaMV35S promoter sequences flanking the CP sequence and had been introduced into C1738 plum. To determine if this structure was enough to induce silencing, cross-hybridization was made with the C1738 clone and the CP expressing but PPV-susceptible C2 clone. In total, 4 out of 63 clones were silenced. While introduction of the IRSH is reduced due to the heterozygous character in C1738 plum, the silencing induced by the IRSH PPV CP is robust. Extensive studies, in greenhouse containment, demonstrated that the genetic resource of C1738 clone can silence the CP production. In addition, these were verified through the virus transgene pyramiding in the BO70146 BlueByrd cv. plum that successfully produced resistant BlueByrd BO70146 × C1738 (HybC1738) hybrid plums.

Defining the 'HoneySweet' insertion event utilizing NextGen sequencing and a de novo genome assembly of plum (Prunus domestica).

'HoneySweet' plum (Prunus domestica) is resistant to Plum pox potyvirus, through an RNAi-triggered mechanism. Determining the precise nature of the transgene insertion event has been complicated due to the hexaploid genome of plum. DNA blots previously indicated an unintended hairpin arrangement of the Plum pox potyvirus coat protein gene as well as a multicopy insertion event. To confirm the transgene arrangement of the insertion event, 'HoneySweet' DNA was subjected to whole genome sequencing using Illumina short-read technology. Results indicated two different insertion events, one containing seven partial copies flanked by putative plum DNA sequence and a second with the predicted inverted repeat of the coat protein gene driven by a double 35S promoter on each side, flanked by plum DNA. To determine the locations of the two transgene insertions, a phased plum genome assembly was developed from the commercial plum 'Improved French'. A subset of the scaffolds (2447) that were >10 kb in length and representing, >95% of the genome were annotated and used for alignment against the 'HoneySweet' transgene reads. Four of eight matching scaffolds spanned both insertion sites ranging from 157,704 to 654,883 bp apart, however we were unable to identify which scaffold(s) represented the actual location of the insertion sites due to potential sequence differences between the two plum cultivars. Regardless, there was no evidence of any gene(s) being interrupted as a result of the insertions. Furthermore, RNA-seq data verified that the insertions created no new transcriptional units and no dramatic expression changes of neighboring genes.

Thermal-responsive genetic and epigenetic regulation of DAM cluster controlling dormancy and chilling requirement in peach floral buds.

The Dormancy-associated MADS-box (DAM) gene cluster in peach serves as a key regulatory hub on which the seasonal temperatures act and orchestrate dormancy onset and exit, chilling response and floral bud developmental pace. Yet, how different temperature regimes interact with and regulate the six linked DAM genes remains unclear. Here, we demonstrate that chilling downregulates DAM1 and DAM3-6 in dormant floral buds with distinct patterns and identify DAM4 as the most abundantly expressed one. We reveal multiple epigenetic events, with tri-methyl histone H3 lysine 27 (H3K27me3) induced by chilling specifically in DAM1 and DAM5, a 21-nt sRNA in DAM3 and a ncRNA induced in DAM4. Such induction is inversely correlated with downregulation of their cognate DAMs. We also show that the six DAMs were hypermethylated, associating with the production of 24-nt sRNAs. Hence, the chilling-responsive dynamic of the different epigenetic elements and their interactions likely define distinct expression abundance and downregulation pattern of each DAM. We further show that the expression of the five DAMs remains steadily unchanged or continuously downregulated at the ensuing warm temperature after chilling, and this state of regulation correlates with robust increase of sRNA expression, H3K27me3 and CHH methylation, which is particularly pronounced in DAM4. Such robust increase of repressive epigenetic marks may irreversibly reinforce the chilling-imposed repression of DAMs to ensure flower-developmental programming free from any residual DAM inhibition. Taken together, we reveal novel information about genetic and epigenetic regulation of the DAM cluster in peach, which will be of fundamental significance in understanding of the regulatory mechanisms underlying chilling requirement and dormancy release, and of practical application for improvement of plasticity of flower time and bud break in fruit trees to adapt changing climates.

Identification of early fruit development reference genes in plum.

An RNAseq study of early fruit development and stone development in plum, Prunus domestica, was mined to identify sets of genes that could be used to normalize expression studies in early fruit development. The expression values of genes previously identified from Prunus as reference genes were first extracted and found to vary considerably in endocarp tissue relative to whole fruit tissue. Nine other genes were chosen that varied less than 2-fold amongst the 20 RNAseq libraries of early fruit development and endocarp tissues. These gene were tested on a series of developmental plum fruit samples to determine if any could be used as a reference gene in the analyses of fruit-based tissues in plum. The three most stable genes as determined using RefFinder were IPGD (imidazole glycerol-phosphate dehydratase), HAM1 (histone acetyltransferase) and SNX1 (sorting nexin 1). These were further tested to analyze genes expressed differentially in endocarp tissue between normal and minimal endocarp cultivars. To determine the universality of those nine genes as fruit development reference genes, three other data sets of RNAseq from peach and apple were analyzed to determine the reference gene expression. Multiple genes exhibited tissue specific patterns of expression while one gene, the SNX1, emerged as possessing a universal pattern between the Rosaceae species, at all developmental stages, and tissue types tested. The results suggest that the use of existing RNAseq data to identify standard genes can provide stable reference genes for a specific tissues or experimental conditions under exploration.

Prophylactic Pancreatectomies Carry Prohibitive Mortality at Low-Volume Centers: A California Cancer Registry Study.

BACKGROUND: Pancreatectomy for malignancy is associated with improved outcomes when performed at high-volume centers. The goal of this study was to assess pancreatectomy outcomes for premalignant cystic lesions as a function of hospital volume. METHODS: The Healthcare Cost and Utilization Project (HCUP) was queried for all pancreatectomies performed in California from 2003 to 2011. Cases were stratified, separating benign versus malignant disease. Hospitals were categorized as low-volume (≤25 pancreatectomies/year; LV) or high-volume (>25; HV) centers. Perioperative morbidity, mortality, and length of stay were compared in HV vs. LV centers. RESULTS: There were 7554 pancreatectomies performed in 201 hospitals during the study period, where 5652 (75%) procedures were performed for malignancy, 338 (4%) for chronic pancreatitis, and 1564 (21%) for benign/premalignant cysts. The majority of pancreatectomies for cystic disease were performed at LV centers (65%). There were no significant differences in length of stay (7 vs. 8 days; p = 0.6) or 90-day readmission rates (12.8% vs. 12.9%; p = 1.0) in HV versus LV centers. However, there were higher surgical (46.2% LV vs. 41.1% HV, p = 0.05) and medical (13.3% LV vs. 9.2% HV; p = 0.017) complications at LV centers. Most importantly, there was a fourfold higher in-hospital mortality at LV centers (2.36% vs. 0.55%; p = 0.007). CONCLUSION: Pancreatic resection for benign lesions at HV hospitals is associated with significantly lower morbidity and mortality, suggesting that when feasible, patients should seek care at high-volume centers for these semi-elective surgeries.

Gibberellic acid induced parthenocarpic 'Honeycrisp' apples (Malus domestica) exhibit reduced ovary width and lower acidity.

Fruit set and development are dependent on auxin, gibberellin, and cytokinin, which cause parthenocarpic development in many species when applied ectopically. Commercial sprays containing these hormones are used to improve apple fruit set, size, and shape, but have been implicated negatively in other aspects of fruit quality. We applied gibberellic acid (GA3), synthetic auxin (NAA), and the auxin-transport inhibitor NPA to 'Honeycrisp' apple flowers. Fruit retention and size were quantified throughout development, and seed number and fruit quality parameters were measured at maturity. GA3 alone caused the development of seedless parthenocarpic apples. At maturity, GA3-treated apples were narrower due to reduced ovary width, indicating that GA3 induced normal growth of the hypanthium, but not the ovary. GA3-treated fruits were also less acidic than hand-pollinated controls, but had similar firmness, starch, and sugar content. To further understand the regulation of parthenocarpy, we performed tissue-specific transcriptome analysis on GA3-treated, NAA-treated, and control fruits, at 18 days after treatment and again at maturity. Overall, transcriptome analysis showed GA3-treated and hand-pollinated fruits were highly similar in RNA expression profiles. Early expression differences in putative cell division, cytokinin degradation, and cell wall modification genes in GA3-treated ovaries correlated with the observed shape differences, while early expression differences in the acidity gene Ma1 may be responsible for the changes in pH. Taken together, our results indicate that GA3 triggers the development of parthenocarpic apple fruit with morphological deviations that correlate with a number of candidate gene expression differences.

Multilocation comparison of fruit composition for 'HoneySweet', an RNAi based plum pox virus resistant plum.

'HoneySweet', a transgenic plum (Prunus domestica) resistant to plum pox virus through RNAi, was deregulated in the U.S. in 2011. The compositional study of 'HoneySweet' fruit was expanded to include locations outside of the US as well as utilizing a wide variety of comparators and different collection years to see the variability possible. The results revealed that plums have a wide variation in composition and that variation among locations was greater than variation among cultivars. This was also the case for different years at one location. The results supported the supposition that the transgene and insertion event had no significant effect on the composition of 'HoneySweet' fruit even under virus pressure, and that it fell in the normal range of composition of commercially grown plums. It also suggested that the effect of environment is as great as that of genetics on the fruit composition of plums.

Genetic characterization of worldwide Prunus domestica (plum) germplasm using sequence-based genotyping.

Prunus domestica commonly known as European plum is a hexaploid fruit tree species cultivated around the world. Locally it is used for fresh consumption, in jams or jellies, and the production of spirits while commercially the fruit is primarily sold dried (prunes). Despite its agricultural importance and long history of cultivation, many questions remain about the origin of this species, the relationships among its many pomological types, and its underlying genetics. Here, we used a sequence-based genotyping approach to characterize worldwide plum germplasm including the potential progenitor Eurasian plum species. Analysis of 405 DNA samples established a set of four clades consistent with the pomological groups Greengages, Mirabelles, European plums, and d'Agen (French) prune plums. A number of cultivars from each clade were identified as likely clonal selections, particularly among the "French" type prune germplasm that is widely cultivated today. Overall, there was relatively low genetic diversity across all cultivated plums suggesting they have been largely inbred and/or derived from a limited number of founders. The results agree with P. domestica having originated as an interspecific hybrid of a diploid P. cerasifera and a tetraploid P. spinosa that itself may have been an interspecific hybrid of P. cerasifera and an unknown Eurasian plum species. The low genetic diversity and lack of true wild-types coupled with the known cultivation history of Eurasian plums imply that P. domestica may have been a product of inter-specific cross breeding and artificial selection by early agrarian Eurasian societies.

Surgical Intervention in Gastric Carcinoid is Associated With Improved Survival in Local and Regional Disease.

INTRODUCTION: Gastric carcinoid is a rare entity with complex management options. This study aims to determine if surgical intervention in patients with local, regional, and metastatic gastric carcinoid is associated with prolonged survival. MATERIALS AND METHODS: The California Cancer Registry merged with the California Office of Statewide Health Planning and Development was queried for patients with a diagnosis of gastric carcinoid (2000 to 2011). Clinicopathologic characteristics, management, and outcomes were evaluated. RESULTS: There were 1012 patients with a diagnosis of gastric carcinoid identified. The median age was 63 (range, 18 to 99) and the majority of patients were women (615, 60.7%). Most patients had localized disease (644, 64%), whereas 9.4% (95) had regional and 13.4% (133) had distant metastases at diagnosis. The majority of patients underwent gastric surgery (56.7%, n=574 vs. 43.2%, n=438). Prolonged survival was associated with gastric surgery in patients with both local (median survival not reached; P<0.0001) and regional disease (27 mo with surgery vs. 5 mo with no surgery; P=0.0007). In patients who underwent gastrectomy and resection of hepatic metastasis, the survival approached those patients who had surgery for only regional disease (26 vs. 27 mo, P=0.8721). CONCLUSIONS: Although the biology of the disease is the most significant predictor of overall outcome, when technically feasible and where comorbidities allow, aggressive endoscopic or surgical intervention should be offered for local and locoregional diseases, respectively.

Exposure in vitro to an Environmentally Isolated Strain TC09 of Cladosporium sphaerospermum Triggers Plant Growth Promotion, Early Flowering, and Fruit Yield Increase.

A growing number of bacteria and fungi have been found to promote plant growth through mutualistic interactions involving elements such as volatile organic compounds (VOCs). Here, we report the identification of an environmentally isolated strain of Cladosporium sphaerospermum (herein named TC09), that substantially enhances plant growth after exposure in vitro beyond what has previously been reported. When cultured on Murashige and Skoog (MS) medium under in vitro conditions, tobacco seedlings (Nicotiana tabacum) exposed to TC09 cultures for 20 days increased stem height and whole plant biomass up to 25- and 15-fold, respectively, over controls without exposure. TC09-mediated growth promotion required >5 g/L sucrose in the plant culture medium and was influenced by the duration of exposure ranging from one to 10 days, beyond which no differences were detected. When transplanted to soil under greenhouse conditions, TC09-exposed tobacco plants retained higher rates of growth. Comparative transcriptome analyses using tobacco seedlings exposed to TC09 for 10 days uncovered differentially expressed genes (DEGs) associated with diverse biological processes including cell expansion and cell cycle, photosynthesis, phytohormone homeostasis and defense responses. To test the potential efficacy of TC09-mediated growth promotion on agricultural productivity, pepper plants (Capsicum annuum L.) of two different varieties, Cayenne and Minisweet, were pre-exposed to TC09 and planted in the greenhouse to monitor growth, flowering, and fruit production. Results showed that treated pepper plants flowered 20 days earlier and yielded up to 213% more fruit than untreated controls. Altogether the data suggest that exposure of young plants to C. sphaerospermum produced VOCs may provide a useful tool to improve crop productivity.

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.

Spatial and temporal assessment of pollen- and seed-mediated gene flow from genetically engineered plum Prunus domestica.

Pollen flow from a 0.46 ha plot of genetically engineered (GE) Prunus domestica located in West Virginia, USA was evaluated from 2000-2010. Sentinel plum trees were planted at distances ranging from 132 to 854 m from the center of the GE orchard. Plots of mixed plum varieties and seedlings were located at 384, 484 and 998 m from the GE plot. Bee hives (Apis mellifera) were dispersed between the GE plum plot and the pollen flow monitoring sites. Pollen-mediated gene flow from out of the GE plum plot to non-GE plums under the study conditions was low, only occurring at all in 4 of 11 years and then in only 0.31% of the 12,116 seeds analyzed. When it occurred, gene flow, calculated as the number of GUS positive embryos/total embryos sampled, ranged from 0.215% at 132 m from the center of the GE plum plot (28 m from the nearest GE plum tree) to 0.033-0.017% at longer distances (384-998 m). Based on the percentage of GUS positive seeds per individual sampled tree the range was 0.4% to 12%. Within the GE field plot, gene flow ranged from 4.9 to 39%. Gene flow was related to distance and environmental conditions. A single year sample from a sentinel plot 132 m from the center of the GE plot accounted for 65% of the total 11-year gene flow. Spatial modeling indicated that gene flow dramatically decreased at distances over 400 m from the GE plot. Air temperature and rainfall were, respectively, positively and negatively correlated with gene flow, reflecting the effects of weather conditions on insect pollinator activity. Seed-mediated gene flow was not detected. These results support the feasibility of coexistence of GE and non-GE plum orchards.

A relational model for spiritually-sensitive hospice care.

When faced with terminal illness, it is natural for hospice patients to question the meaning of life. Hospice workers need to have the ability to assist patients in dealing with these questions in case patients need their assistance. Helping patients deal with questions about life meaning is associated with spiritual care. The following article presents a qualitative study on the provision of spiritual care by hospice workers. The results are used to inform a relational model for spiritually-sensitive hospice care that demonstrates how a variety of individual factors have the potential to influence the delivery of spiritual care.

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.

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.

BACKGROUND: MicroRNAs (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. RESULTS: We 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. CONCLUSIONS: Collectively, 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.

Stone formation in peach fruit exhibits spatial coordination of the lignin and flavonoid pathways and similarity to Arabidopsis dehiscence.

BACKGROUND: Lignification of the fruit endocarp layer occurs in many angiosperms and plays a critical role in seed protection and dispersal. This process has been extensively studied with relationship to pod shatter or dehiscence in Arabidopsis. Dehiscence is controlled by a set of transcription factors that define the fruit tissue layers and whether or not they lignify. In contrast, relatively little is known about similar processes in other plants such as stone fruits which contain an extremely hard lignified endocarp or stone surrounding a single seed. RESULTS: Here we show that lignin deposition in peach initiates near the blossom end within the endocarp layer and proceeds in a distinct spatial-temporal pattern. Microarray studies using a developmental series from young fruits identified a sharp and transient induction of phenylpropanoid, lignin and flavonoid pathway genes concurrent with lignification and subsequent stone hardening. Quantitative polymerase chain reaction studies revealed that specific phenylpropanoid (phenylalanine ammonia-lyase and cinnamate 4-hydroxylase) and lignin (caffeoyl-CoA O-methyltransferase, peroxidase and laccase) pathway genes were induced in the endocarp layer over a 10 day time period, while two lignin genes (p-coumarate 3-hydroxylase and cinnamoyl CoA reductase) were co-regulated with flavonoid pathway genes (chalcone synthase, dihydroflavanol 4-reductase, leucoanthocyanidin dioxygen-ase and flavanone-3-hydrosylase) which were mesocarp and exocarp specific. Analysis of other fruit development expression studies revealed that flavonoid pathway induction is conserved in the related Rosaceae species apple while lignin pathway induction is not. The transcription factor expression of peach genes homologous to known endocarp determinant genes in Arabidopsis including SHATTERPROOF, SEEDSTCK and NAC SECONDARY WALL THICENING PROMOTING FACTOR 1 were found to be specifically expressed in the endocarp while the negative regulator FRUITFUL predominated in exocarp and mesocarp. CONCLUSIONS: Collectively, the data suggests, first, that the process of endocarp determination and differentiation in peach and Arabidopsis share common regulators and, secondly, reveals a previously unknown coordination of competing lignin and flavonoid biosynthetic pathways during early fruit development.