Suppression Subtractive Hybridization Analysis of Genes Regulated by Application of Exogenous Abscisic Acid in Pepper Plant (Capsicum annuum L.) Leaves under Chilling Stress

Low temperature is one of the major factors limiting pepper (Capsicum annuum L.) production during winter and early spring in non-tropical regions. Application of exogenous abscisic acid (ABA) effectively alleviates the symptoms of chilling injury, such as wilting and formation of necrotic lesions on pepper leaves; however, the underlying molecular mechanism is not understood. The aim of this study was to identify genes that are differentially up- or downregulated in ABA-pretreated hot pepper seedlings incubated at 6°C for 48 h, using a suppression subtractive hybridization (SSH) method. A total of 235 high-quality ESTs were isolated, clustered and assembled into a collection of 73 unigenes including 18 contigs and 55 singletons. A total of 37 unigenes (50.68%) showed similarities to genes with known functions in the non-redundant database; the other 36 unigenes (49.32%) showed low similarities or unknown functions. Gene ontology analysis revealed that the 37 unigenes could be classified into nine functional categories. The expression profiles of 18 selected genes were analyzed using quantitative RT-PCR; the expression levels of 10 of these genes were at least two-fold higher in the ABA-pretreated seedlings under chilling stress than water-pretreated (control) plants under chilling stress. In contrast, the other eight genes were downregulated in ABA-pretreated seedlings under chilling stress, with expression levels that were one-third or less of the levels observed in control seedlings under chilling stress. These results suggest that ABA can positively and negatively regulate genes in pepper plants under chilling stress.     

不同低温需求量砂梨休眠相关基因PpMADS13的特征及表达模式

为探究福建主栽的不同低温需求量砂梨Pyrus pyrifolia品种中休眠相关基因PpMADS13-1和PpMADS13-3序列及表达特征,以‘黄花’、‘蜜雪’两品种休眠时期的花芽为材料,克隆PpMADS13-1和PpMADS13-3基因,利用荧光定量PCR技术分析基因在休眠时期的表达变化情况。结果显示,PpMADS13-1序列在砂梨两品种间较保守,而PpMADS13-3序列在两品种间保守性较差;‘黄花’梨PpMADS13-1hh和PpMADS13-3hh在休眠期间随着休眠的解除下调表达,‘蜜雪’梨PpMADS13-1mx和PpMADS13-3mx在休眠期间呈现“M”型表达趋势。PpMADS13-1和PpMADS13-3均对砂梨的花芽休眠解除起到调控作用,在不同低温需求量品种间PpMADS13-1和PpMADS13-3的休眠调控模式有所不同。     

Clinal variation of dormancy progression in apricot

The aim of this study was to determine the bud dormancy progression in apricot at different latitudes and altitudes. Six locations in regions with a Mediterranean climate in South Africa (SA) and Spain were chosen. The study was carried out during two consecutive years, 2007 and 2008, in SA and results were compared to those obtained in Spain in 2008. Locations ranged from low-chill areas, such as Ladismith and Villiersdorp in SA and Campotéjar in Spain, to high-chill areas, such as Ceres in SA and Barranda in Spain. A number of apricot cultivars comprising the range of chilling requirements in both countries were selected. In addition, a second, parallel study was performed to evaluate the paradormancy progression in ‘Palsteyn’ (SA) and ‘Rojo Pasión’ (Spain). Deeper dormancy was not observed in high-chill cultivars located in cold areas than in low-chill cultivars in warm areas. However, low-chill cultivars located in warm areas entered and released from dormancy earlier than high chill cultivars in warm areas. Thus, a clinal variation in dormancy progression under warm temperatures in apricot cultivars is suggested. The role of photoperiod and minimum temperatures is proposed to have a key role in dormancy onset. Paradoxically, an earlier maximum depth of dormancy was found in those areas with higher minimum temperatures at the end of summer. Before the beginning of winter, all cultivars showed an important increase of budburst rate, which indicated the end of endodormancy. Afterwards an ecodormancy period followed during winter, while chilling continued to accumulate. These results contrast with the assumed concept of the breaking of dormancy through chilling accumulation during winter and suggest a possible mediation by photoperiod in overcoming of dormancy. On the other hand, paradormancy exerted a reduction in budburst rate during dormancy entry, whereas decapitation increased the budburst rate throughout the dormant season, indicating interaction between different plant parts during this period.     

Diapause termination of Rhagoletis cerasi pupae is regulated by local adaptation and phenotypic plasticity: escape in time through bet‐hedging strategies

Persistence and thriving of univoltine, herbivore insect species of the temperate zone rely on obligate diapause response that ensures winter survival and synchronization with host phenology. We used a stenophagous fruit fly (Rhagoletis cerasi) with obligate pupae diapause to determine genetic and environmental effects on diapause intensity of geographically isolated populations with habitat heterogeneity. Pupae from two Greek and one German populations with various gene flow rates were exposed at five constant chilling temperatures (0–12 °C) for different durations and then incubated at a high temperature until all adults have emerged. Pupae diapause intensity differs among Greek and German populations, suggesting an adaptive response to habitat heterogeneity (mostly differences in phenology patterns of local host cultivars). Moderately warm winter temperatures, such as 8 °C, promote diapause termination in all three populations. Insufficient chilling (short duration or warmer temperatures) regulates the expression of prolonged dormancy. Interestingly, extended chilling (longer than required for terminating diapause) ‘return’ pupae to another (facultative) cycle of dormancy enabling adults to emerge during the next appropriate ‘window of time’; a strategy first time reported for univoltine insects. Consequently, diapause duration of R. cerasi is determined both by i) the adaptive response to local climatic conditions (annual dormancy) and ii) the plastic responses to interannual climatic variability resulting in two types of long life cycles within populations, prolonged and facultative dormancy as response to insufficient chilling and extended exposure to chilling, respectively. Long life cycles are expressed as a part of dormancy bet‐hedging strategies of R. cerasi populations.     

Performance and long-term stability of the barley hordothionin gene in multiple transgenic apple lines

Introduction of sustainable scab resistance in elite apple cultivars is of high importance for apple cultivation when aiming at reducing the use of chemical crop protectants. Genetic modification (GM) allows the rapid introduction of resistance genes directly into high quality apple cultivars. Resistance genes can be derived from apple itself but genetic modification also opens up the possibility to use other, non-host resistance genes. A prerequisite for application is the long-term performance and stability of the gene annex trait in the field. For this study, we produced and selected a series of transgenic apple lines of two cultivars, i.e. ‘Elstar’ and ‘Gala’ in which the barley hordothionin gene (hth) was introduced. After multiplication, the GM hth-lines, non-GM susceptible and resistant controls and GM non-hth controls were planted in a random block design in a field trial in 40 replicates. Scab resistance was monitored after artificial inoculation (first year) and after natural infection (subsequent years). After the trial period, the level of expression of the hth gene was checked by quantitative RT-PCR. Four of the six GM hth apple lines proved to be significantly less susceptible to apple scab and this trait was found to be stable for the entire 4-year period. Hth expression at the mRNA level was also stable.     

Dissection of chilling requirement and bloom date QTLs in peach using a whole genome sequencing of sibling trees from an F2 mapping population

Chilling requirement (CR) for floral bud dormancy release is one of the major limiting factors for geographical adaptation of fruiting trees. Using a whole genome sequencing approach (Illumina platform), we explored polymorphism underlying phenotypic differences among individuals in a peach F₂ cross segregating for chilling requirement and bloom date. Allelic configuration of individuals, which represented phenotypic extremes in the cross (300 vs. 1,100 chill hours) allowed reconstruction of low- and high-chill haplotypes within three most significant quantitative trait locus (QTL) intervals on the Prunus G1, G4, and G7. We detected single nucleotide polymorphic sites (SNPs), small deletions and insertions (DIPs), and large structural variants (SVs) associated with low-chill haplotypes and created a prioritized list of candidate genes based on functionally characterized homologs from Arabidopsis thaliana. Two dormancy associated genes PpeDAM5 and PpeDAM6 are the strongest candidate genes for the major QTL signal at the lower end of G1. Also, key functional genes involved in the Polycomb repressive mechanism, cell cycle progression, and hormone regulation were evident as strong candidate genes underlying QTL intervals in this peach cross.     

Transcript Profiling of Paoenia ostii during Artificial Chilling Induced Dormancy Release Identifies Activation of GA Pathway and Carbohydrate Metabolism

Endo-dormant flower buds must pass through a period of chilling to reinitiate growth and subsequent flowering, which is a major obstacle to the forcing culture of tree peony in winter. Customized cDNA microarray (8×15 K element) was used to investigate gene expression profiling in tree peony ‘Feng Dan Bai’ buds during 24 d chilling treatment at 0–4°C. According to the morphological changes after the whole plants were transferred to green house, endo-dormancy was released after 18 d chilling treatment, and prolonged chilling treatment increased bud break rate. Pearson correlation hierarchical clustering of sample groups was highly consistent with the dormancy transitions revealed by morphological changes. Totally 3,174 significantly differentially-expressed genes (P<0.05) were observed through endo-dormancy release process, of which the number of up-regulated (1,611) and that of down-regulated (1,563) was almost the same. Functional annotation of differentially-expressed genes revealed that cellular process, metabolic process, response to stimulus, regulation of biological process and development process were well-represented. Hierarchical clustering indicated that activation of genes involved in carbohydrate metabolism (Glycolysis, Citrate cycle and Pentose phosphate pathway), energy metabolism and cell growth. Based on the results of GO analysis, totally 51 probes presented in the microarray were associated with GA response and GA signaling pathway, and 22 of them were differently expressed. The expression profiles also revealed that the genes of GA biosynthesis, signaling and response involved in endo-dormancy release. We hypothesized that activation of GA pathway played a central role in the regulation of dormancy release in tree peony.     

Molecular characterization of cisgenic lines of apple ‘Gala’ carrying the Rvi6 scab resistance gene

Using resistance genes from a crossable donor to obtain cultivars resistant to diseases and the use of such cultivars in production appears an economically and environmentally advantageous approach. In apple, introgression of resistance genes by classical breeding results in new cultivars, while introducing cisgenes by biotechnological methods maintains the original cultivar characteristics. Recently, plants of the popular apple ‘Gala’ were genetically modified by inserting the apple scab resistance gene Rvi6 (formerly HcrVf2) under control of its own regulatory sequences. This gene is derived from the scab‐resistant apple ‘Florina’ (originally from the wild apple accession Malus floribunda 821). The vector used for genetic modification allowed a postselection marker gene elimination to achieve cisgenesis. In this work, three cisgenic lines were analysed to assess copy number, integration site, expression level and resistance to apple scab. For two of these lines, a single insertion was observed and, despite a very low expression of 0.07‐ and 0.002‐fold compared with the natural expression of ‘Florina’, this was sufficient to induce plant reaction and reduce fungal growth by 80% compared with the scab‐susceptible ‘Gala’. Similar results for resistance and expression analysis were obtained also for the third line, although it was impossible to determine the copy number and TDNA integration site–such molecular characterization is requested by the (EC) Regulation No. 1829/2003, but may become unnecessary if cisgenic crops become exempt from GMO regulation.     

Relative developmental, environmental, and tree-to-tree variability in buds from field-grown apple trees

High-throughput genomic technologies are becoming more accessible to nonmodel plant species, and therefore, tissue collected outside controlled environments is being increasingly used for microarray analyses. In this study, we present a 15,720-feature apple microarray analysis of the variability of gene expression in buds from field-grown apple trees. Tree-to-tree and day-to-day variances were assessed during two different seasons: summer, when the meristems in the buds were undergoing the first stages of floral development, and autumn, when the buds were undergoing transition to winter dormancy. We found that apple trees with the same scion and rootstock cultivars, grown in a standard orchard environment, had small tree-to-tree variation. Gene expression differences caused by season was the dominant cause of variance while using false discovery rate control with a threshold of α* = 0.01 to select significantly different expression between genes. At this threshold, the environmental and location effects accounted for less than 10% of the genes selected. Consequently, we have shown that field microarray experiments are a viable approach for measuring seasonal changes in gene expression during apple bud development. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

ParSOC1, a MADS-box gene closely related to Arabidopsis AGL20/SOC1, is expressed in apricot leaves in a diurnal manner and is linked with chilling requirements for dormancy break

Dormancy break is a physiological phenomenon associated with the ability of plants to cope with changing environmental conditions and adjust their growth habits accordingly. In order to understand the potential role of genes in the control of dormancy break ParSOC1, a distinct apricot MADS-box gene which is most closely related to the AGL20/SOC1 MADS-box family was studied in several apricot cultivars that differ in their chilling requirements. The ParSOC1 gene is expressed in a diurnal manner and is highly polymorphic among apricot cultivars in the transcribed region upstream to the putative ATG translation initiation site. Genotyping of 48 different apricot cultivars revealed 13 different ParSOC1 alleles. By associating the chilling requirements of the apricot cultivars with their ParSOC1 genotype, it was possible to demonstrate a significant correlation between the presence of specific ParSOC1 alleles and chilling requirements. The data provided suggest that ParSOC1 or a gene in its close proximity could be involved in the regulation of dormancy break of vegetative shoots in apricot.     

Biochemical characterisation of MdCXE1, a carboxylesterase from apple that is expressed during fruit ripening

Esters are an important component of apple (Malus × domestica) flavour. Their biosynthesis increases in response to the ripening hormone ethylene, but their metabolism by carboxylesterases (CXEs) is poorly understood. We have identified 16 members of the CXE multigene family from the commercial apple cultivar, ‘Royal Gala’, that contain all the conserved features associated with CXE members of the α/β hydrolase fold superfamily. The expression of two genes, MdCXE1 and MdCXE16 was characterised in an apple fruit development series and in a transgenic line of ‘Royal Gala’ (AO3) that is unable to synthesise ethylene in fruit. In wild-type MdCXE1 is expressed at low levels during early stages of fruit development, rising to a peak of expression in apple fruit at harvest maturity. It is not significantly up-regulated by ethylene in the skin of AO3 fruit. MdCXE16 is expressed constitutively in wild-type throughout fruit development, and is up-regulated by ethylene in skin of AO3 fruit. Semi-purified recombinant MdCXE1 was able to hydrolyse a range of 4-methyl umbelliferyl ester substrates that included those containing acyl moieties that are found in esters produced by apple fruit. Kinetic characterisation of MdCXE1 revealed that the enzyme could be inhibited by organophosphates and that its ability to hydrolyse esters showed increasing affinity (K_m) but decreasing turnover (k_cat) as substrate acyl carbon length increases from C2 to C16. Our results suggest that MdCXE1 may have an impact on apple flavour through its ability to hydrolyse relevant flavour esters in ripe apple fruit.     

Mapping of Candidate Genes Involved in Bud Dormancy and Flowering Time in Sweet Cherry (Prunus avium)

The timing of flowering in perennial plants is crucial for their survival in temperate climates and is regulated by the duration of bud dormancy. Bud dormancy release and bud break depend on the perception of cumulative chilling during endodormancy and heat during the bud development. The objectives of this work were to identify candidate genes involved in dormancy and flowering processes in sweet cherry, their mapping in two mapping progenies ‘Regina’ × ‘Garnet’ and ‘Regina’ × ‘Lapins’, and to select those candidate genes which co-localized with quantitative trait loci (QTLs) associated with temperature requirements for bud dormancy release and flowering. Based on available data on flowering processes in various species, a list of 79 candidate genes was established. The peach and sweet cherry orthologs were identified and primers were designed to amplify sweet cherry candidate gene fragments. Based on the amplified sequences of the three parents of the mapping progenies, SNPs segregations in the progenies were identified. Thirty five candidate genes were genetically mapped in at least one of the two progenies and all were in silico mapped. Co-localization between candidate genes and QTLs associated with temperature requirements and flowering date were identified for the first time in sweet cherry. The allelic composition of the candidate genes located in the major QTL for heat requirements and flowering date located on linkage group 4 have a significant effect on these two traits indicating their potential use for breeding programs in sweet cherry to select new varieties adapted to putative future climatic conditions.     

Transcript profiling in Vitis riparia during chilling requirement fulfillment reveals coordination of gene expression patterns with optimized bud break

Endodormant grapevine buds require a period of chilling before they break and begin to grow. Custom Vitis bud cDNA microarrays (9,216 features) were used to examine gene expression patterns in overwintering Vitis riparia buds during 2,000 h of 4°C chilling. Three-node cuttings collected concurrently with buds were monitored to determine dormancy status. Chilling requirement was fulfilled after 1,500 h of chilling; however, 2,000 h of chilling significantly increased the rate of bud break. Microarray analysis identified 1,469 significantly differentially expressed (p value < 0.05) array features when 1,000, 1,500, and 2,000 h of chilling were compared to 500 h of chilling. Functional classification revealed that the majority of genes were involved in metabolism, cell defense/stress response, and genetic information processing. The number of significantly differentially expressed genes increased with chilling hour accumulation. The expression of a group of 130 genes constantly decreased during the chilling period. Up-regulated genes were not detected until the later stages of chilling accumulation. Hierarchical clustering of non-redundant expressed sequence tags revealed inhibition of genes involved in carbohydrate and energy metabolism and activation of genes involved in signaling and cell growth. Clusters with expression patterns associated with increased chilling and bud break were identified, indicating several candidate genes that may serve as indicators of bud chilling requirement fulfillment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.