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.

Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus 'Robusta 5' accessions.

BACKGROUND: Breeding of fire blight resistant scions and rootstocks is a goal of several international apple breeding programs, as options are limited for management of this destructive disease caused by the bacterial pathogen Erwinia amylovora. A broad, large-effect quantitative trait locus (QTL) for fire blight resistance has been reported on linkage group 3 of Malus 'Robusta 5'. In this study we identified markers derived from putative fire blight resistance genes associated with the QTL by integrating further genetic mapping studies with bioinformatics analysis of transcript profiling data and genome sequence databases. RESULTS: When several defined E.amylovora strains were used to inoculate three progenies from international breeding programs, all with 'Robusta 5' as a common parent, two distinct QTLs were detected on linkage group 3, where only one had previously been mapped. In the New Zealand 'Malling 9' X 'Robusta 5' population inoculated with E. amylovora ICMP11176, the proximal QTL co-located with SNP markers derived from a leucine-rich repeat, receptor-like protein (MxdRLP1) and a closely linked class 3 peroxidase gene. While the QTL detected in the German 'Idared' X 'Robusta 5' population inoculated with E. amylovora strains Ea222_JKI or ICMP11176 was approximately 6 cM distal to this, directly below a SNP marker derived from a heat shock 90 family protein gene (HSP90). In the US 'Otawa3' X 'Robusta5' population inoculated with E. amylovora strains Ea273 or E2002a, the position of the LOD score peak on linkage group 3 was dependent upon the pathogen strains used for inoculation. One of the five MxdRLP1 alleles identified in fire blight resistant and susceptible cultivars was genetically associated with resistance and used to develop a high resolution melting PCR marker. A resistance QTL detected on linkage group 7 of the US population co-located with another HSP90 gene-family member and a WRKY transcription factor previously associated with fire blight resistance. However, this QTL was not observed in the New Zealand or German populations. CONCLUSIONS: The results suggest that the upper region of 'Robusta 5' linkage group 3 contains multiple genes contributing to fire blight resistance and that their contributions to resistance can vary depending upon pathogen virulence and other factors. Mapping markers derived from putative fire blight resistance genes has proved a useful aid in defining these QTLs and developing markers for marker-assisted breeding of fire blight resistance.

Identification of genes differentially expressed during interaction of resistant and susceptible apple cultivars (Malus x domestica) with Erwinia amylovora.

BACKGROUND: The necrogenic enterobacterium, Erwinia amylovora is the causal agent of the fire blight (FB) disease in many Rosaceae species, including apple and pear. During the infection process, the bacteria induce an oxidative stress response with kinetics similar to those induced in an incompatible bacteria-plant interaction. No resistance mechanism to E. amylovora in host plants has yet been characterized, recent work has identified some molecular events which occur in resistant and/or susceptible host interaction with E. amylovora: In order to understand the mechanisms that characterize responses to FB, differentially expressed genes were identified by cDNA-AFLP analysis in resistant and susceptible apple genotypes after inoculation with E. amylovora. RESULTS: cDNA were isolated from M.26 (susceptible) and G.41 (resistant) apple tissues collected 2 h and 48 h after challenge with a virulent E. amylovora strain or mock (buffer) inoculated. To identify differentially expressed transcripts, electrophoretic banding patterns were obtained from cDNAs. In the AFLP experiments, M.26 and G.41 showed different patterns of expression, including genes specifically induced, not induced, or repressed by E. amylovora. In total, 190 ESTs differentially expressed between M.26 and G.41 were identified using 42 pairs of AFLP primers. cDNA-AFLP analysis of global EST expression in a resistant and a susceptible apple genotype identified different major classes of genes. EST sequencing data showed that genes linked to resistance, encoding proteins involved in recognition, signaling, defense and apoptosis, were modulated by E. amylovora in its host plant. The expression time course of some of these ESTs selected via a bioinformatic analysis has been characterized. CONCLUSION: These data are being used to develop hypotheses of resistance or susceptibility mechanisms in Malus to E. amylovora and provide an initial categorization of genes possibly involved in recognition events, early signaling responses the subsequent development of resistance or susceptibility. These data also provided potential candidates for improving apple resistance to fire blight either by marker-assisted selection or genetic engineering.

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 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.

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.

Genome-wide identification and analysis of the SBP-box family genes in apple (Malus × domestica Borkh.).

SQUAMOSA promoter binding protein (SBP)-box genes encode a family of plant-specific transcription factors and play many crucial roles in plant development. In this study, 27 SBP-box gene family members were identified in the apple (Malus × domestica Borkh.) genome, 15 of which were suggested to be putative targets of MdmiR156. Plant SBPs were classified into eight groups according to the phylogenetic analysis of SBP-domain proteins. Gene structure, gene chromosomal location and synteny analyses of MdSBP genes within the apple genome demonstrated that tandem and segmental duplications, as well as whole genome duplications, have likely contributed to the expansion and evolution of the SBP-box gene family in apple. Additionally, synteny analysis between apple and Arabidopsis indicated that several paired homologs of MdSBP and AtSPL genes were located in syntenic genomic regions. Tissue-specific expression analysis of MdSBP genes in apple demonstrated their diversified spatiotemporal expression patterns. Most MdmiR156-targeted MdSBP genes, which had relatively high transcript levels in stems, leaves, apical buds and some floral organs, exhibited a more differential expression pattern than most MdmiR156-nontargeted MdSBP genes. Finally, expression analysis of MdSBP genes in leaves upon various plant hormone treatments showed that many MdSBP genes were responsive to different plant hormones, indicating that MdSBP genes may be involved in responses to hormone signaling during stress or in apple development.

Electroporation and stable maintenance of plasmid DNAs in a biocontrol strain of Pseudomonas syringae.

Transformation efficiencies as high as 10(7) transformants microg(-1) DNA have been previously reported for pseudomonads using electroporation protocols established for E. coli with plasmid DNAs prepared from methylation proficient E. coli hosts. We report here a protocol for electroporation of plasmid DNAs into a biocontrol strain of Pseudomonas syringae which could not be electroporated by standard E. coli methods. Transformation efficiencies of 10(7) or higher were obtained with DNA recovered from initial P. syringae transformation or with DNA prepared from methylation deficient E. coli. Both plasmids used in this study were stably maintained in the absence of selection for at least 50 generations.

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.