The relationship of cold acclimation and extracellular ice formation to winter thermonasty in two Rhododendron species and their F1 hybrid.

PREMISE: Thermonastic leaf movements in evergreen Rhododendron species have been used to study plant strategies for winter photoprotection. To add to the current fundamental understanding of this behavior, we addressed the following questions: (1) Is the cold-acclimated (CA) state necessary for thermonasty, and do cold-induced leaf movements also occur in non-acclimated (NA) plants? (2) Which of the two movements, leaf rolling versus curling, is more responsive to freezing, if any, in a non-thermonastic species? (3) What is the temporal relationship between extracellular freezing and thermonasty? (4) What genetic inferences can be drawn from leaf movement in an F1 hybrid relative to its parents? METHODS: A temperature-controlled, gradual cooling regime was used to quantify freeze-induced leaf movements. Infrared thermography was used to confirm extracellular ice-formation in leaves. RESULTS: Both NA and CA plants of thermonastic species exhibited thermonasty, but leaf rolling/curling increased significantly in CA plants. In the cold-acclimated condition, a non-thermonastic species showed almost no rolling during freezing, while the thermonastic species and F1 hybrid did, the latter exhibiting a response intermediate to the parents. Freezing-induced leaf curling in the non-thermonastic species and the F1 hybrid was equivalent and significantly less than the degree of curling in the thermonastic species. CONCLUSIONS: Milder thermonasty in NA than CA leaves could be associated with differential anisotropy in the rolling forces and/or response of aquaporins to freezing. Leaf movements in the hybrid suggest that leaf rolling and curling are additive and dominant genetic traits, respectively. Infrared thermography confirms that ice formation in tissues precedes cold-induced thermonasty in R. catawbiense.

Biocontrol activity of a cold-adapted yeast from Tibet against gray mold in cherry tomato and its action mechanism.

Cold-adapted biocontrol yeast was selected from four yeast isolates from Tibet against gray mold of cherry tomato in cold storage. The strain numbered LB2 showed the best biocontrol activity and identified as Cryptococcus laurentii. Competition for nutrient, space, and induced fruit resistance was also its antagonistic mechanism. Compared with C. laurentii from sea-level place, the reason why LB2 had a better biocontrol activity was studied. More trehalose and proline in cell of LB2 made it exhibit a better cellular activity at low temperature, such as higher population dynamics in the wounds of cherry tomato and more biocontrol-related enzyme secretion, chitinase and ß-glucanase. The better oxidative stress tolerance was another characteristic of LB2. Maybe because of the ideal culture condition, there was no obvious difference between these two yeasts in the growth in vitro test at low temperature. Although the same phenomenon existed in the low pH stress test, LB2 still had higher cell concentration under this stress. Comparative transcriptomics method was also applied to analyze the cell activity of LB2 and C. laurentii at different temperatures. The results showed that more active response in the intracellular structure and intracellular metabolic process to cold temperature made LB2 had a better activity. The present study indicated a possibility to select cold-adapted biocontrol yeast from Tibet and also showed its primary action mechanism.

Fire Blight Resistance in Wild Accessions of Malus sieversii.

Fire blight (Erwinia amylovora) is a devastating bacterial disease in apple that results in severe economic losses. Epidemics are becoming more common as susceptible cultivars and rootstocks are being planted, and control is becoming more difficult as antibiotic-resistant strains develop. Resistant germplasm currently being utilized by breeding programs tend to have small fruit size and poor flavor characteristics. Malus sieversii, a progenitor species of domestic apple, is notable for its relatively large, palatable fruit and some accessions have been reported to be resistant to fire blight. In this study, nearly 200 accessions of M. sieversii and appropriate controls were inoculated with E. amylovora in both Washington and West Virginia to identify fire blight resistant accessions. Twelve accessions were identified with resistance comparable to highly resistant and resistant controls. Several accessions exhibited a unique resistance response, not previously reported in domestic apple (M. × domestica), characterized by low incidence of infection but high severity once infection was initiated. Several of these M. sieversii accessions will be used as parents in future crosses in the Washington State University apple breeding program.

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.

Genes responding to water deficit in apple (Malus × domestica Borkh.) roots.

BACKGROUND: Individual plants adapt to their immediate environment using a combination of biochemical, morphological and life cycle strategies. Because woody plants are long-lived perennials, they cannot rely on annual life cycle strategies alone to survive abiotic stresses. In this study we used suppression subtractive hybridization to identify genes both up- and down-regulated in roots during water deficit treatment and recovery. In addition we followed the expression of select genes in the roots, leaves, bark and xylem of 'Royal Gala' apple subjected to a simulated drought and subsequent recovery. RESULTS: In agreement with studies from both herbaceous and woody plants, a number of common drought-responsive genes were identified, as well as a few not previously reported. Three genes were selected for more in depth analysis: a high affinity nitrate transporter (MdNRT2.4), a mitochondrial outer membrane translocase (MdTOM7.1), and a gene encoding an NPR1 homolog (MpNPR1-2). Quantitative expression of these genes in apple roots, bark and leaves was consistent with their roles in nutrition and defense. CONCLUSIONS: Additional genes from apple roots responding to drought were identified using suppression subtraction hybridization compared to a previous EST analysis from the same organ. Genes up- and down-regulated during drought recovery in roots were also identified. Elevated levels of a high affinity nitrate transporter were found in roots suggesting that nitrogen uptake shifted from low affinity transport due to the predicted reduction in nitrate concentration in drought-treated roots. Suppression of a NPR1 gene in leaves of drought-treated apple trees may explain in part the increased disease susceptibility of trees subjected to dehydrative conditions.

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.

Identification of differentially expressed genes associated with changes in the morphology of Pichia fermentans on apple and peach fruit.

Pichia fermentans (strain DISAABA 726) is an effective biocontrol agent against Monilinia fructicola and Botrytis cinerea when inoculated in artificially wounded apple fruit but is an aggressive pathogen when inoculated on wounded peach fruit, causing severe fruit decay. Pichia fermentans grows as budding yeast on apple tissue and exhibits pseudohyphal growth on peach tissue, suggesting that dimorphism may be associated with pathogenicity. Two complementary suppressive subtractive hybridization (SSH) strategies, that is, rapid subtraction hybridization (RaSH) and PCR-based subtraction, were performed to identify genes differentially expressed by P. fermentans after 24-h growth on apple vs. peach fruit. Gene products that were more highly expressed on peach than on apple tissue, or vice versa, were sequenced and compared with available yeast genome sequence databases. Several of the genes more highly expressed, when P. fermentans was grown on peach, were related to stress response, glycolysis, amino acid metabolism, and alcoholic fermentation but surprisingly not to cell wall degrading enzymes such as pectinases or cellulases. The dual activity of P. fermentans as both a biocontrol agent and a pathogen emphasizes the need for a thorough risk analysis of potential antagonists to avoid unpredictable results that could negatively impact the safe use of postharvest biocontrol strategies.

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.

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.