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.

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.

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.

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.

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.

Overdiagnosis of bipolar disorder among substance use disorder inpatients with mood instability.

BACKGROUND: Among substance use disorder (SUD) patients, mood instability and high-risk behaviors may suggest the presence of bipolar disorder. However, active substance abuse impedes efforts to diagnose bipolar illness validly in patients with mood complaints. METHOD: The authors retrospectively reviewed records for 85 adults admitted sequentially over a 1-year period (August 1, 2005, to July 31, 2006) to a private inpatient dual-diagnosis unit for substance abuse/dependence and mood disorders. A senior research psychiatrist conducted diagnostic interviews based on DSM-IV criteria to ascertain current and lifetime manic or hypomanic episodes during abstinent periods. RESULTS: Only 33% of subjects with suspected bipolar diagnoses (28/85) met DSM-IV criteria for bipolar I or II disorder. DSM-IV bipolar patients were significantly older (p = .029) and more likely to have made past suicide attempts (p = .027), abused fewer substances (p = .027), and were less likely to abuse cocaine (p < .001) than those failing to meet DSM-IV criteria. Inability to affirm bipolar diagnoses most often resulted from insufficient DSM-IV "B" symptoms associated with mania or hypomania (55% or 45/82), inability to identify abstinent periods for assessing mood symptoms (36%, 29/81), and inadequate durations of manic/hypomanic symptoms for DSM-IV syndromic criteria (12%, 10/84). Patients not meeting DSM-IV criteria were most often presumed to have bipolar disorder solely on the basis of the presence of mood instability, although this feature held little predictive value for DSM-IV bipolar diagnoses. CONCLUSIONS: Many patients with active SUDs who are diagnosed in the community with bipolar disorder may not actually meet DSM-IV criteria for bipolar I or II disorder. Caution must be exercised when attempting to diagnose such patients, particularly when mood instability or cocaine use is present.

A minimal peach type II chlorophyll a/b-binding protein promoter retains tissue-specificity and light regulation in tomato.

BACKGROUND: Promoters with tissue-specificity are desirable to drive expression of transgenes in crops to avoid accumulation of foreign proteins in edible tissues/organs. Several photosynthetic promoters have been shown to be strong regulators of expression of transgenes in light-responsive tissues and would be good candidates for leaf and immature fruit tissue-specificity, if expression in the mature fruit were minimized. RESULTS: A minimal peach chlorophyll a/b-binding protein gene (Lhcb2*Pp1) promoter (Cab19) was isolated and fused to an uidA (beta-glucuronidase [GUS]) gene containing the PIV2 intron. A control vector carrying an enhanced mas35S CaMV promoter fused to uidA was also constructed. Two different orientations of the Cab19::GUS fusion relative to the left T-DNA border of the binary vector were transformed into tomato. Ten independent regenerants of each construct and an untransformed control line were assessed both qualitatively and quantitatively for GUS expression in leaves, fruit and flowers, and quantitatively in roots. CONCLUSION: The minimal CAB19 promoter conferred GUS activity primarily in leaves and green fruit, as well as in response to light. GUS activity in the leaves of both Cab19 constructs averaged about 2/3 that observed with mas35S::GUS controls. Surprisingly, GUS activity in transgenic green fruit was considerably higher than leaves for all promoter constructs; however, in red, ripe fruit activities were much lower for the Cab19 promoter constructs than the mas35S::GUS. Although GUS activity was readily detectable in flowers and roots of mas35S::GUStransgenic plants, little activity was observed in plants carrying the Cab19 promoter constructs. In addition, the light-inducibility of the Cab19::GUS constructs indicated that all the requisite cis-elements for light responsiveness were contained on the Cab19 fragment. The minimal Cab19 promoter retains both tissue-specificity and light regulation and can be used to drive expression of foreign genes with minimal activity in mature, edible fruit.

Developmental regulation of peach ACC oxidase promoter--GUS fusions in transgenic tomato fruits.

A genomic DNA sequence (PpACO1) encoding 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) from peach (Prunus persica L. Batsch cv. Loring) was isolated. It has four exons interrupted by three introns and 2.9 kb of flanking region 5' of the translational start codon. Previous work with the cDNA demonstrated that accumulation of the peach ACO message correlated with increasing amounts of ethylene synthesized by the fruit as they ripened. To identify regulatory elements in the peach ACC oxidase gene, chimeric fusions between 403, 610, 901, 1319, 2141, and 2919 bp of the 5' flanking region of the PpACO1 sequence and the beta-glucuronidase (GUS) coding sequence were constructed and used to transform tomato (Lycopersicon esculentum [Mill] cv. Pixie). Fruits from the various promoter lines were analysed for GUS expression by histochemical GUS staining, GUS quantitative enzyme activity determination, and measuring the relative amounts of GUS mRNA. Constructs with the smallest promoter of 403 bp had significant GUS expression in fruit, but not in other tissues, indicating the presence of a region that affects tissue-specific expression. An increase in GUS expression was observed with promoters longer than 901 bp, indicating an enhancer region between -1319 and -901. The full-length promoter of 2919 bp directed GUS expression in the green stage of fruit development, and increased GUS expression as fruit matured, indicating a regulatory region between -2919 and -2141 that controls the temporal expression of the gene in fruit. Only the full-length promoter sequence demonstrated responsiveness to ethylene.

Deletions in an endopolygalacturonase gene cluster correlate with non-melting flesh texture in peach.

Endopolygalacturonase activity during softening of peach [Prunus persica (L.) Batsch] fruit is thought to be responsible for the melting flesh texture. A cDNA, PRF5, was previously identified as a fruit-related endopolygalacturonase that may be involved with the texture differences (Lester et al. 1996). We found that all eight of the non-melting flesh cultivars in this study had a deletion in at least one of their PRF5-related polygalacturonase genes, while none of the melting flesh cultivars did. There were three sources of the non-melting trait, as identified by the extent of the deletions. One source of non-melting flesh resulted in a complete deletion of PRF5-related genes while the other two sources had deletions of a subset of those genes, suggesting a cluster of polygalacturonase genes at the melting flesh locus. All of the non-melting flesh cultivars used for this study had greatly reduced or undetectable mRNA levels of PRF5-related polygalacturonase during fruit softening. Using PCR techniques, it was determined that either the PRF5 gene, or more likely, an unidentified peach polygalacturonase at the same locus, is responsible for melting flesh texture determination.

Characterization of a type II chlorophyll a/b-binding protein gene (Lhcb2*Pp1) in peach. II. mRNA abundance in developing leaves exposed to sun or shade.

Leaf development of shoots exposed to full sunlight and shoots shaded by the canopy was followed in field-grown, mature peach trees (Prunus persica (L.) Batsch, cv. Loring) during the first half of the 1995 growing season. The architecture and size of shaded shoots and sun-exposed shoots differed significantly. Total number of leaves produced on shaded shoots was significantly less than on sun-exposed shoots throughout the season, and differences in leaf number between light conditions increased as the season progressed. The overall patterns of leaf development along sun-exposed and shaded shoots were qualitatively similar. The expression pattern of the type II chlorophyll a/b-binding protein gene, Lhcb2*Pp1, determined by RNA abundance in leaves at different positions along the shoot, was also similar between the two light conditions. The major difference between sun-exposed and shaded leaves was a lower abundance of Lhcb2*Pp1 RNA in mature, shaded leaves compared with sun-exposed leaves. Although the number of fruit per shoot was significantly lower on shaded shoots than on sun-exposed shoots, the rate of fruit drop was not substantially different during the growing season, indicating that quantitative differences in leaf initiation and growth caused by differences in light exposure did not adversely affect fruit retention. However, based on comparison with a previous study of leaf development in non-fruiting trees, reproductive development slowed the rate of vegetative growth without affecting the overall pattern of leaf development along the shoots.

Induction of Chitinase, beta-1,3-Glucanase, and Phenylalanine Ammonia Lyase in Peach Fruit by UV-C Treatment.

ABSTRACT Treatment of peach fruit with UV-C light caused a rapid induction of chitinase, beta-1,3-glucanase, and phenylalanine ammonia lyase (PAL) activities starting 6 h after treatment and reaching maximum levels at 96 h after treatment. By 96 h after UV-C treatment, chitinase, beta-1,3-glucanase, and PAL activities in UV-C-treated fruit were over twofold above the levels observed for the control. In nontreated control fruit, no apparent increase in chitinase and beta-1,3-glucanase activities was detected but a minor increase in PAL activity was seen. The transient increase in chitinase, beta-1,3-glucanase, and PAL activities in UV-C-treated fruit was preceded by a gradual activation of the corresponding genes. UV-C-treated fruit showed an increase in accumulation of beta-1,3-glucanase and chitinase mRNAs at 3 h after treatment, which peaked approximately 96 h posttreatment. A similar induction kinetic pattern was observed for PAL mRNA in response to UV-C treatment, except the induction started 6 h after UV-C treatment. These results show that the response of peach fruit to elicitor treatment is similar to that seen in other plant-elicitors interactions and suggests the involvement of peach biochemical defense responses in UV-C-mediated disease resistance.

Characterization of the peach homologue of the ethylene receptor, PpETR1, reveals some unusual features regarding transcript processing.

To identify which processes in peach, Prunus persica [L.] Batsch., are associated with changes in ethylene perception, we cloned and characterized a peach homologue of the gene encoding the ethylene receptor, ETR1. A fragment of the peach gene, PpETR1, obtained via PCR using degenerate primers against peach genomic DNA was used to screen a cDNA library made from ripening fruit. The resulting cDNA and subsequent 3' RACE clones indicate that the PpETR1 coding region is highly similar to other ETR1 homologues. However, the mRNA undergoes unusual alternative splicing that potentially results in three different mature transcripts. Use of an alternative 3' splice site to remove the last intron in PpETR1a results in a polypeptide that is missing three amino acids within the receiver-like domain. Retention of the terminal intron occurs in PpETR1b, which, if translated, would result in a truncated protein lacking a receiver-like domain. Fruit from three cultivars with substantially different ripening times were examined from 7 to 130 days after bloom using RT-PCR to characterize expression of the intron-retaining and fully spliced mRNAs. There were only slight differences in the abundance of these mRNAs among cultivars during fruit development; however, one of the slow-ripening cultivars showed a substantial increase in expression of the unspliced mRNA in pre-climacteric fruit. Variations in PpETR1 transcript abundance in wounding experiments indicate that the properly spliced and unspliced versions have different accumulation patterns in fruit, whereas both are essentially constitutive in leaves. These observations indicate that changes in ethylene sensitivity may occur during wounding in fruit.