Apple CRISPR-Cas9-A Recipe for Successful Targeting of AGAMOUS-like Genes in Domestic Apple.

Fruit trees and other fruiting hardwood perennials are economically valuable, and there is interest in developing improved varieties. Both conventional breeding and biotechnology approaches are being utilized towards the goal of developing advanced cultivars. Increased knowledge of the effectiveness and efficiency of biotechnology approaches can help guide use of the CRISPR gene-editing technology. Here, we examined CRISPR-Cas9-directed genome editing in the valuable commodity fruit tree Malus x domestica (domestic apple). We transformed two cultivars with dual CRISPR-Cas9 constructs designed to target two AGAMOUS-like genes simultaneously. The main goal was to determine the effectiveness of this approach for achieving target gene changes. We obtained 6 Cas9 control and 38 independent CRISPR-Cas9 events. Of the 38 CRISPR-Cas9 events, 34 (89%) had gene edits and 14 (37%) showed changes to all alleles of both target genes. The most common change was large deletions, which were present in 59% of all changed alleles, followed by small deletions (21%), small insertions (12%), and a combination of small insertions and deletions (8%). Overall, a high rate of successful gene alterations was found. Many of these changes are predicted to cause frameshifts and alterations to the predicted peptides. Future work will include monitoring the floral development and floral form.

From endodormancy to ecodormancy: the transcriptional landscape of apple floral buds.

This study endeavors to explore the transcriptomic profiles of two apple cultivars, namely, 'Honeycrisp' and 'Cripps Pink,' which represent late and early-blooming cultivars, respectively. Using RNA-sequencing technology, we analyzed floral bud samples collected at five distinct time intervals during both endodormancy and ecodormancy. To evaluate the transcriptomic profiles of the 30 sequenced samples, we conducted principal component analysis (PCA). PC1 explained 43% of the variance, separating endodormancy and ecodormancy periods, while PC2 explained 16% of the variance, separating the two cultivars. The number of differentially expressed genes (DEGs) increased with endodormancy progression and remained elevated during ecodormancy. The majority of DEGs were unique to a particular time point, with only a few overlapping among or between the time points. This highlights the temporal specificity of gene expression during the dormancy transition and emphasizes the importance of sampling at multiple time points to capture the complete transcriptomic dynamics of this intricate process. We identified a total of 4204 upregulated and 7817 downregulated DEGs in the comparison of endodormancy and ecodormancy, regardless of cultivar, and 2135 upregulated and 2413 downregulated DEGs in the comparison of 'Honeycrisp' versus 'Cripps Pink,' regardless of dormancy stage. Furthermore, we conducted a co-expression network analysis to gain insight into the coordinated gene expression profiles across different time points, dormancy stages, and cultivars. This analysis revealed the most significant module (ME 14), correlated with 1000 GDH and consisting of 1162 genes. The expression of the genes within this module was lower in 'Honeycrisp' than in 'Cripps Pink.' The top 20 DEGs identified in ME 14 were primarily related to jasmonic acid biosynthesis and signaling, lipid metabolism, oxidation-reduction, and transmembrane transport activity. This suggests a plausible role for these pathways in governing bud dormancy and flowering time in apple.

Ethylene-Mediated Modulation of Bud Phenology, Cold Hardiness, and Hormone Biosynthesis in Peach (Prunus persica).

Spring frosts exacerbated by global climate change have become a constant threat to temperate fruit production. Delaying the bloom date by plant growth regulators (PGRs) has been proposed as a practical frost avoidance strategy. Ethephon is an ethylene-releasing PGR found to delay bloom in several fruit species, yet its use is often coupled with harmful effects, limiting its applicability in commercial tree fruit production. Little information is available regarding the mechanisms by which ethephon influences blooming and bud dormancy. This study investigated the effects of fall-applied ethephon on bud phenology, cold hardiness, and hormonal balance throughout the bud dormancy cycle in peach. Our findings concluded that ethephon could alter several significant aspects of peach bud physiology, including accelerated leaf fall, extended chilling accumulation period, increased heat requirements, improved cold hardiness, and delayed bloom date. Ethephon effects on these traits were primarily dependent on its concentration and application timing, with a high concentration (500 ppm) and an early application timing (10% leaf fall) being the most effective. Endogenous ethylene levels were induced significantly in the buds when ethephon was applied at 10% versus 90% leaf fall, indicating that leaves are essential for ethephon uptake. The hormonal analysis of buds at regular intervals of chilling hours (CH) and growing degree hours (GDH) also indicated that ethephon might exert its effects through an abscisic acid (ABA)-independent way in dormant buds. Instead, our data signifies the role of jasmonic acid (JA) in mediating budburst and bloom in peach, which also appears to be influenced by ethephon treatment. Overall, this research presents a new perspective in interpreting horticultural traits in the light of biochemical and molecular data and sheds light on the potential role of JA in bud dormancy, which deserves further attention in future studies that aim at mitigating spring frosts.

An apple rootstock overexpressing a peach CBF gene alters growth and flowering in the scion but does not impact cold hardiness or dormancy.

The C-repeat binding factor (CBF) transcription factor is involved in responses to low temperature and water deficit in many plant species. Overexpression of CBF genes leads to enhanced freezing tolerance and growth inhibition in many species. The overexpression of a peach CBF (PpCBF1) gene in a transgenic line of own-rooted apple (Malus×domestica) M.26 rootstock (T166) trees was previously reported to have additional effects on the onset of dormancy and time of spring budbreak. In the current study, the commercial apple cultivar 'Royal Gala' (RG) was grafted onto either non-transgenic M.26 rootstocks (RG/M.26) or transgenic M.26 (T166) rootstocks (RG/T166) and field grown for 3 years. No PpCBF1 transcript was detected in the phloem or cambium of RG scions grafted on T166 rootstocks indicating that no graft transmission of transgene mRNA had occurred. In contrast to own-rooted T166 trees, no impact of PpCBF1 overexpression in T166 rootstocks was observed on the onset of dormancy, budbreak or non-acclimated leaf-cold hardiness in RG/T166 trees. Growth, however, as measured by stem caliper, current-year shoot extension and overall height, was reduced in RG/T166 trees compared with RG/M.26 trees. Although flowering was evident in both RG/T166 and RG/M.26 trees in the second season, the number of trees in flower, the number of shoots bearing flowers, and the number of flower clusters per shoot was significantly higher in RG/M.26 trees than RG/T166 trees in both the second and third year after planting. Elevated levels of RGL (DELLA) gene expression were observed in RG/T166 trees and T166 trees, which may play a role in the reduced growth observed in these tree types. A model is presented indicating how CBF overexpression in a rootstock might influence juvenility and flower abundance in a grafted scion.

Engineering carpel-specific cold stress tolerance: a case study in Arabidopsis.

Climate change predictions forecast an increase in early spring frosts that could result in severe damage to perennial crops. For example, the Easter freeze of April 2007 left several states in the United States reporting a complete loss of that year's peach crop. The most susceptible organ to early frost damage in fruit trees is the carpel, particularly during bloom opening. In this study, we explored the use of a carpel-specific promoter (ZPT2-10) from petunia (Petunia hybrida var. Mitchell) to drive expression of the peach dehydrin PpDhn1. In peach, this gene is exceptionally responsive to low temperature but has not been observed to be expressed in carpels. This study examined carpel-specific properties of a petunia promoter driving the expression of the GUS gene (uidA) in transgenic Arabidopsis flowers and developed a carpel-specific ion leakage test to assess freezing tolerance. A homozygous Arabidopsis line (line 1-20) carrying the petunia ZPT2-10 promoter::PpDhn1 construct was obtained and freezing tolerance in the transgenic line was compared with an untransformed control. Overexpression of PpDhn1 in line 1-20 provided as much as a 1.9°C increase in carpel freezing tolerance as measured by electrolyte leakage.

CBF gene expression in peach leaf and bark tissues is gated by a circadian clock.

CBF (C-repeat Binding Factor) transcription factors are part of the AP2/ERF (Apetala2-ethylene responsive factor) domain family of DNA-binding proteins that recognize a C-repeat response cis-acting element that regulates a number of cold-responsive genes (CBF regulon). Induction of CBF gene expression by low temperature in Arabidopsis has been shown to be gated by a circadian clock. In peach (Prunus persica L.), five CBF genes are arranged in tandem on scaffold (linkage group) 5 of the peach genome. Since CBF gene regulation has been shown to be more complex in woody plants than herbaceous plants, the present study was conducted to determine if temperature-modulated CBF gene expression in peach leaf and bark tissues was also influenced by a circadian clock. One-year-old 'Loring' peach trees grafted on 'Bailey' rootstocks were entrained to a 12-h day/12-h night photoperiod at 25 °C. After 2 weeks, trees were exposed to 4 °C under continuous light for up to 48 h beginning at either subjective dawn + 4 h (ZT4; where ZT is Zeitgeber time) or subjective dawn + 16 h (ZT16) with leaf and bark tissues harvested at various time points. Gene expression of the five peach CBF genes and a DREB2 gene was assessed by real-time quantitative polymerase chain reaction. Results revealed a distinct gating of CBF gene expression by a circadian clock for four CBF genes in both leaf and bark tissues. CBF genes were highly induced by 4 °C in ZT4 leaf samples with expression peaking at 6-24 h depending on the specific CBF gene. In contrast, CBF gene expression was highly attenuated in leaf, and to a lesser extent in bark, samples exposed to 4 °C at ZT16. These results are similar to reports for Arabidopsis. Further experiments were conducted to verify environmental influence on the induction of CBF and DREB2 genes. In contrast to DREB2 genes from other dicots, the peach DREB2 ortholog was induced by both low temperature and dehydration. Induction of the peach CBFs and DREB2 by either low temperature or dehydration corresponded with regulatory motifs present in their promoter sequences. Low temperature and dehydration induction data for three peach dehydrin genes indicated that the regulation of these genes in peach is complex, with individual dehydrin gene expression being correlated with the expression of one or more CBF genes.

Comparative expression and transcript initiation of three peach dehydrin genes.

Dehydrin genes encode proteins with demonstrated cryoprotective and antifreeze activity, and they respond to a variety of abiotic stress conditions that have dehydration as a common component. Two dehydrins from peach (Prunus persica L. [Batsch.]) have been previously characterized; here, we describe the characterization of a third dehydrin from peach bark, PpDhn3, isolated by its response to low temperature. The expression of all three dehydrin genes was profiled by semi-quantitative reverse transcription PCR, and transcript initiation was mapped for all three genes using the RNA ligase-mediated 5' rapid amplification of cDNA ends technique. PpDhn3 transcripts from bark collected in December or July, as well as transcripts from developing fruit, initiated at a single site. Although most of the PpDhn1 transcripts initiated at a similar position, those from young fruit initiated much further upstream of the consensus TATA box. Bark and fruit transcripts encoding PpDhn2 initiated ca. 30 bases downstream of a consensus TATA box; however, transcripts from ripe fruit initiated further upstream. Ripe fruit transcripts of PpDhn2 contain a 5' leader intron which is predicted to add some 34 amino acids to the N-terminal methionine of the cognate protein when properly processed. Secondary structure prediction of sequences surrounding the TATA box suggests that conformational transitions associated with decreasing temperature contribute to the regulation of expression of the cold-responsive dehydrin genes. Taken together these results reveal new, unexpected levels of gene regulation contributing to the overall expression pattern of peach dehydrins.

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.

Differential regulation of two dehydrin genes from peach (Prunus persica) by photoperiod, low temperature and water deficit.

Dehydrins are one of several proteins that have been specifically associated with qualitative and quantitative changes in cold hardiness. Recent evidence indicates that the regulation of dehydrin genes by low nonfreezing temperature (LT) and short photoperiod (SD) can be complex and deserves more detailed analysis to better understand the role of specific dehydrin genes and proteins in the response of woody plants to environmental stress. We have identified a new peach (Prunus persica (L.) Batsch) dehydrin gene (PpDhn2) and examined the responses of this gene and a previously identified dehydrin (PpDhn1) to SD, LT and water deficit. PpDhn2 was strongly induced by water deficit but not by LT or SD. It was also present in the mature embryos of peach. In contrast, PpDhn1 was induced by water deficit and LT but not by SD. We conducted an in silico analysis of the promoters of these genes and found that the promoter region of PpDhn1 contained two dehydration-responsive-elements (DRE)/C-repeats that are responsive to LT and several abscisic acid (ABA)-response elements (ABREs). In contrast, the promoter region of PpDhn2 contained no LT elements but contained several ABREs and an MYCERD1 motif. Both promoter analyses were consistent with the observed expression patterns. The discrepancy between field-collected samples and growth-chamber experiments in the expression of PpDhn1 in response to SD suggests that SD-induced expression of dehydrin genes is complex and may be the result of several interacting factors.

Characterization of an S-locus receptor protein kinase-like gene from peach.

A receptor-like protein kinase gene (Ppsrkl1) was isolated from a peach (Prunus persica (L.) Batsch.) bark cDNA library prepared with RNAs isolated from bark collected in December (cold acclimated). Sequence analysis indicated that this gene is related to the S-locus family of receptor protein kinases (SRKs) and that it shares greatest homology with ZMPK1 from maize and At4g32300 from Arabidopsis, both of which are intron-less genes. In bark tissues, Ppsrkl1 is induced by water deficit treatment, repressed by short-day photoperiods and showed no response to cold treatment. The Ppsrkl1 mRNA also increased in roots in response to water deficit. In fruit, Ppsrkl1 shows no response up to 6 h after wounding, but at 12 and 24 h after wounding, Ppsrkl1 mRNA shows an abrupt decline. This decline was prevented by the addition of salicylic acid to the wound site. The Ppsrkl1 mRNA rapidly decreased in fruit after 10-min exposure to UV-C radiation, followed by a return to normal levels within 1.5 h. Taken together, these experiments indicate that Ppsrkl1 is negatively regulated by light and positively influenced by salicylic acid treatment in fruit and water stress in bark and roots.

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.