Expansion and evolutionary patterns of GDSL-type esterases/lipases in Rosaceae genomes

GDSL-type esterase/lipase (GELP) is mainly characterized by a conserved GDSL domain at N terminus, and is widely found in all living species, both prokaryotes and eukaryotes. GELP gene family consists of a wide range of members playing important roles in plant physiological processes, such as development, stress responses, and functional divergences. In our study, 597 GELP genes were identified from six Rosaceae genomes (i.e., Fragaria vesca, Prunus persica, Prunus avium, Prunus mume, Pyrus bretschneideri, and Malus domestica) by a comprehensive analysis. All GELP genes were further divided into ten subfamilies based on phylogenetic tree analysis. Subfamily D and subfamily E are the two largest subfamilies. Microcollinearity analysis suggested that WGD/segmental events contribute to the expansion of the GELP gene family in M. domestica and P. bretschneideri compared to F. vesca, P. persica, P. avium, and P. mume. Some PbGELPs were expressed during the fruit development of P. bretschneideri and pollen tubes, indicating their activity in these tissues. The expression divergence of PbGELP duplication gene pairs suggests that many mutations were allowed during evolution, although the structure of GELP genes was highly conserved. The current study results provided the feasibility to understand the expansion and evolution patterns of GELP in Rosaceae genomes, and highlight the function during P. bretschneideri fruits and pollen tubes development.     

Doubled haploid production in fruit crops

The interest of fruit breeders in haploids and doubled haploids (DH), lies in the possibility of shortening the time needed to produce homozygous lines compared to conventional breeding. Haplo-diploidization through gametic embryogenesis allows single-step development of complete homozygous lines from heterozygous parents. In a conventional breeding programme, a pure line is developed after several generations of selfing. With fruit crops, characterized by a long reproductive cycle, a high degree of heterozygosity, large size, and, sometimes, self-incompatibility, there is no way to obtain haploidization through conventional methods. This paper reviews the current status of research on doubled haploid production in the main fruit crops: Citrus, Malus domestica, Pyrus communis, Pyrus pyrifolia, Prunus persica, Prunus avium, Prunus domestica, Prunus armeniaca, Vitis vinifera, Actinidia deliciosa, Olea europaea, Morus alba, Actinidia deliziosa, [Musa balbisiana (BB)], Carica papaya, Annona squamosa, Feijoa sellowiana, Opuntia ficus-indica, Eriobotrya japonica.     

Study of 'Redhaven' peach and its white-fleshed mutant suggests a key role of CCD4 carotenoid dioxygenase in carotenoid and norisoprenoid volatile metabolism

Background  

Carotenoids are plant metabolites which are not only essential in photosynthesis but also important quality factors in determining the pigmentation and aroma of flowers and fruits. To investigate the regulation of carotenoid metabolism, as related to norisoprenoids and other volatile compounds in peach (Prunus persica L. Batsch.), and the role of carotenoid dioxygenases in determining differences in flesh color phenotype and volatile composition, the expression patterns of relevant carotenoid genes and metabolites were studied during fruit development along with volatile compound content. Two contrasted cultivars, the yellow-fleshed 'Redhaven' (RH) and its white-fleshed mutant 'Redhaven Bianca' (RHB) were examined.     

Host preference of the plum curculio

We assessed host preference of adult plum curculio, Conotrachelus nenuphar (Herbst) (Coleoptera: Curculionidae), based on the total number of mark‐released and wild adults recovered and the total distance moved by mark‐released adults in an orchard whose layout was designed to specifically allow foraging plum curculios to choose among host tree species. Host trees included apple, Malus domestica Borkh.; pear, Pyrus communis (L.); peach, Prunus persica (L.) Batsch; apricot, Prunus armeniaca L.; tart cherry, Prunus cerasus L.; sweet cherry, Prunus avium (L.); European plum, Prunus domestica L.; and Japanese plum, Prunus salicina Lindl. (all Rosaceae). We released 2900 marked adults and recovered 17.7%. We used screen traps to provide a measure of the number of adults that arrived at and climbed up particular host trees and found that significantly greater numbers of marked adults and the greatest number of wild adults were recovered from screen traps attached to Japanese plum. We sampled host tree canopies by tapping limbs to provide a measure of the number of adults within a tree canopy at a particular moment. Again, significantly greater numbers of marked and wild adults were recovered from plum species, with no difference between Japanese and European plum cultivars for marked individuals, but with significantly greater numbers of wild individuals recovered from Japanese plum. The preference index (PI) for Japanese plum based on total distances moved by all marked adults recovered on Japanese plum divided by the total distance moved by marked adults recovered on other host trees indicated that Japanese plum was the most highly preferred host, followed by European plum, peach, sweet cherry, tart cherry, apricot, apple, and pear, respectively.     

A simple and efficient method for DNA extraction from grapevine cultivars andVitis species

A quick, simple, and reliable method for the extraction of DNA from grapevine species, hybrids, andAmpelopsis brevipedunculata (Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTBA-based extraction procedure modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method has also been used successfully for extraction of total DNA from other fruit species such as apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica), and raspberry (Rubus idaeus). DNA yield from this procedure is high (up to 1 mg/g of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.     

Bioinformatic and expression analyses on carotenoid dioxygenase genes in fruit development and abiotic stress responses in <Emphasis Type="Italic">Fragaria vesca</Emphasis>

Carotenoid dioxygenases, including 9-cis-epoxycarotenoid dioxygenases (NCEDs) and carotenoid cleavage dioxygenases (CCDs), can selectively cleave carotenoids into various apocarotenoid products that play important roles in fleshy fruit development and abiotic stress response. In this study, we identified 12 carotenoid dioxygenase genes in diploid strawberry Fragaria vesca, and explored their evolution with orthologous genes from nine other species. Phylogenetic analyses suggested that the NCED and CCDL groups moderately expanded during their evolution, whereas gene numbers of the CCD1, CCD4, CCD7, and CCD8 groups maintained conserved. We characterized the expression profiles of FveNCED and FveCCD genes during flower and fruit development, and in response to several abiotic stresses. FveNCED1 expression positively responded to osmotic, cold, and heat stresses, whereas FveNCED2 was only induced under cold stress. In contrast, FveNCED2 was the unique gene highly and continuously increasing in receptacle during fruit ripening, which co-occurred with the increase in endogenous abscisic acid (ABA) content previously reported in octoploid strawberry. The differential expression patterns suggested that FveNCED1 and FveNCED2 were key genes for ABA biosynthesis in abiotic stress responses and fruit ripening, respectively. FveCCD1 exhibited the highest expression in most stages of flower and fruit development, while the other FveCCDs were expressed in a subset of stages and tissues. Our study suggests distinct functions of FveNCED and FveCCD genes in fruit development and stress responses and lays a foundation for future study to understand the roles of these genes and their metabolites, including ABA and other apocarotenoid products, in the growth and development of strawberry.     

Influence of selected fruit tree pollen on life history of <Emphasis Type="Italic">Euseius stipulatus</Emphasis> (Acari: Phytoseiidae)

Euseius stipulatus (Athias-Henriot) is a predatory mite widespread in the Mediterranean region considered to be important for the biological control of spider mites in citrus orchards. Development, survival and reproduction of this phytoseiid mite feeding on seven commercially obtained pollen were studied under constant laboratory conditions (20 ± 1°C, RH 65 ± 5%, photoperiod 16L: 8D h). Mites were kept individually at rearing units with ample quantity of almond (Prunus amygdalus Batch), apple (Malus domestica Borkh), apricot (Prunus armeniaca L.), cherry (Prunus avium L.), pear (Pyrus communis L.), plum (Prunus domestica L.) and walnut (Juglans regia L.) pollen as food source. Developmental time from egg to adult varied between the several pollen tested from 8.38 ± 0.08 to 9.58 ± 0.11 days for females and from 8.23 ± 0.12 and 9.07 ± 0.12 days for males. Female longevity varied from 11.53 ± 1.22 to 51.38 ± 2.45 days, while fecundity ranged from 22.84 ± 2.30 to 43.61 ± 3.78 eggs/female. The predator was unable to reproduce when feeding on walnut pollen. Data were submitted to life table analysis and values of the intrinsic rate of increase were derived, ranging from 0.079 to 0.146 (day⁻¹). The cumulative Weibull function that was used to describe the age specific survival of females produced excellent fits to the survival data. Results show that almond, plum, cherry and apricot pollen possess higher nutritional value for E. stipulatus than pear and apple pollen and thus may contribute in sustaining and increasing the predator population in field conditions. Walnut pollen can be utilized by the predator only to survive during short periods of time when principal or alternative food sources are scarce.     

苹果乙烯不敏感基因EIN2片段的克隆及序列分析

分别以苹果果实总DNA和cDNA为模板,采用PCR、RT-PCR方法扩增、克隆乙烯不敏感基因(ethyleneinsensitive 2,EIN2),并利用生物信息学方法分析其核苷酸序列和蛋白质结构。结果表明:(1)以DNA和cDNA为模板的扩增结果完全相同,扩增的EIN2基因片段为4 378bp,尚未发现有内含子,开放阅读框全长3 282bp,编码1 093个氨基酸;苹果EIN2相对分子质量为118.9kD,等电点为5.52,其蛋白可能为脂溶性疏水蛋白。(2)所克隆苹果EIN2基因编码的氨基酸序列与拟南芥(AAD41077.1)、碧桃(ACY78397.1)和葡萄(CAN66374.1)EIN2基因编码的氨基酸序列一致性分别为52%、79%、62%。(3)构建的EIN2基因进化树显示,拟南芥、小盐芥、甜瓜、杨毛果EIN2基因亲缘关系较近,聚为一类;葡萄为一类;蒺藜苜蓿为一类;碧桃、矮牵牛、西红柿聚为一类;苹果单独为一类。而且苹果EIN2基因与碧桃等同源基因的亲缘关系相对较近,与拟南芥、小盐芥同源基因的亲缘关系相对较远。     

Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta

Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.     

Isolation and functional analysis of MdNAS1, with functions in improved iron stress tolerance and abnormal flower in transgenic tobacco

Metal elements are essential micronutrients required by all plants for natural physiological activities. Nicotianamine is considered as the chelate substance in the transport of metal ions. In the present study, a new gene encoding NA synthase was isolated from Malus domestica (L.) Borkh and designated as MdNAS1. The expression levels of MdNAS1 were enriched in leaf, and phloem which were highly affected by Fe stress, indoleacetic acid (IAA) and abscisic acid (ABA) treatments in M. domestica seedlings. Subcellular localization research revealed that MdNAS1 was localized in cytoplasmic membrane. Overexpression of MdNAS1 in transgenic tobaccos increased the tolerance to Fe stress, but also contributes to higher chlorophyll, NA, Fe, Mn, Cu and Zn contents and abnormal flowers. Moreover, the MdNAS1-OE tobaccos had the increased expression levels of Fe uptake and transport related genes (NtFRO, NtIRT1, NtVIT, NtNRAMP1, and NtYSL).     

鸭梨PAL基因的反义遗传转化及表达分析

苯丙氨酸解氨酶(phenylalanin ammonia-lyase,PAL,EC4.3.1.5)是植物通过苯丙烷代谢途径合成木质素的关键酶和限速酶,其通过影响木质素的合成而与果实中石细胞的分化、发育及果实品质密切相关。为了降低鸭梨中苯丙氨酸解氨酶的含量,该研究利用反义PAL基因遗传转化鸭梨、降低鸭梨内源PAL基因的表达。结果表明:(1)采用RT-PCR技术,利用根据Gen Bank中西洋梨PAL基因序列设计特异性引物,扩增得到496 bp的鸭梨PAL基因片段。(2)将扩增片段反向插入载体p BI121的MCS区域,构建植物PAL基因反义表达载体p BI121-As PAL。接着采用电转化法将反义表达载体转入农杆菌EHA105中,并制备出农杆菌工程菌液。(3)利用农杆菌介导法对鸭梨组培苗叶片外植体进行遗传转化,得到23株转基因鸭梨苗。PCR检测证实PAL反义基因片段转入鸭梨中,实时定量PCR检测表明转基因鸭梨苗体内PAL基因表达量均有所降低,为非转基因苗的65%~75%。该研究结果表明利用反义RNA技术获得了抑制内源性PAL基因表达的转基因鸭梨植株,为改善鸭梨果实品质、改良品种奠定了基础。     

Evolutionary analysis of S-RNase genes from Rosaceae species

Eight new cDNA sequences for S-RNases were cloned and analysed from almond (Prunus dulcis) cultivars of European origin, and compared to published sequences from other Rosaceae species. Insertions/deletions of 10-20 amino acid residues were detected in the RC4 and C5 domains of S-RNases from almond and sweet cherry. The S-RNases of the Prunus species and those of the genera Malus and Pyrus formed two distinct groups on phylogenetic analysis. Nucleotide substitutions were analysed in the S-RNase genes of these species. The S-genes of almond and sweet cherry have a lower Ka/Ks value than those of apple, pear and wild apple do. The fact that there is no fixed difference between the S-RNase genes of almond and sweet cherry, or between apple and pear, suggests that nucleotide substitutions only introduce transient polymorphism into the two groups, and rarely became fixed and contribute to divergence. Through the comparative study of 17 S-RNase genes from the genus Prunus and 18 from the genera Malus and Pyrus, some fixed nucleotide differences between the two groups were identified. These differences do not appear to be the result of selection for adaptive mutations, since the number of replacement substitutions is not significantly greater than the number of synonymous substitutions. S-RNase genes of almond and sweet cherry, and of apple and pear, showed little heterogeneity in nucleotide substitution rates. However, heterogeneity was observed between the two groups of S-alleles, with the Prunus alleles exhibiting a lower rate of non-synonymous substitutions than alleles from Malus and Pyrus. The evolutionary relationships between these species are discussed.     

Ovipositional preference ofSiphoninus phillyreae and its fitness on seven host plant species

The relationship between ovipositional preference ofSiphoninus phillyreae (Haliday) (Homoptera: Aleyrodidae) and host plant suitability on seven host plant species (Citrus sinensis (L.) cv. ‘Washington’ [navel orange],Fraxinus uhdei (Wenz.) [shamel ash],Heteromeles arbutifolia Roemer [toyon],Malus domestica Mill. cv. ‘Granny Smith’, [apple],Pistacia vera L. cv. ‘Kerman’ [pistachio],Prunus persica (L.) cv. ‘O’Henry’ [peach], andPyrus communis L. cv. ‘Bartlett’ [pear]) was evaluated. Ovipositional preference ofS. phillyreae was determined by measuring egg density after adult female whitefies were given a simultaneous choice of all host plants for oviposition. Immature survival, developmental time, and adult size were examined to determine host plant suitability forS. phillyreae. All studies were performed under greenhouse conditions.S. phillyreae showed distinct ovipositional preference among host plant species. Host plant species had a significant effect on immature survival, but little or no effect on developmental time or forewing length. For four of the seven host plant species tested, there was an association between ovipositional preference and survival.     

In planta cleavage of carotenoids by <Emphasis Type="Italic">Arabidopsis</Emphasis> carotenoid cleavage dioxygenase 4 in transgenic rice plants

A family of carotenoid cleavage dioxygenases (CCDs) produces diverse apocarotenoid compounds via the oxidative cleavage of carotenoids as substrates. Their types are highly dependent on the action of the CCD family to cleave the double bonds at the specific position on the carotenoids. Here, we report in vivo function of the AtCCD4 gene, one of the nine members of the Arabidopsis CCD gene family, in transgenic rice plants. Using two independent single-copy rice lines overexpressing the AtCCD4 transgene, the targeted analysis for carotenoids and apocarotenoids showed the markedly lowered levels of β-carotene (74 %) and lutein (72 %) along with the changed levels of two β-carotene (C₄₀) cleavage products, a two-fold increase of β-ionone (C₁₃) and de novo generation of β-cyclocitral (C₁₀) at lower levels, compared with non-transgenic rice plants. It suggests that β-carotene could be the principal substrate being cleaved at 9–10 (9′–10′) for β-ionone and 7–8 (7′–8′) positions for β-cyclocitral by AtCCD4. This study is in planta report on the generation of apocarotenal volatiles from carotenoid substrates via cleavage by AtCCD4. We further verified that the production of these volatiles was due to the action of exogenous AtCCD4 and not the expression of endogenous rice CCD genes (OsCCD1, 4a, and 4b).     

Tissue-specific and constitutive expression of a hypothetical gene At2g37610 in transgenic Arabidopsis

Hypothetical genes should play important roles in plant growth and development, although their biological functions await elucidation. One of these genes, namely At2g37610, caught our attention during the gene cloning of several salt-tolerant mutants. Promoter-GUS fusion analysis indicated a unique tissue-specific expression pattern of At2g37610 in Arabidopsis. Constitutive expression of the gene under 35S promoter caused obvious morphological changes in transgenic Arabidopsis plants, such as curled rosette leaves and bushy phenotype at maturity. Phenotypic characterization revealed that the cause of the bushy phenotype was the enhanced lateral bud outgrowth at the bottom region of the primary inflorescence, which is different from that of reported mutant plants (bushy or branched) such as max, axr1, and bus mutants. Together, these data suggest that At2g37610 is a possible novel gene related to the regulation of leaf development and shoot patterning.     

Influence of selected fruit tree pollen on life history of Euseius stipulatus (Acari: Phytoseiidae)

Euseius stipulatus (Athias-Henriot) is a predatory mite widespread in the Mediterranean region considered to be important for the biological control of spider mites in citrus orchards. Development, survival and reproduction of this phytoseiid mite feeding on seven commercially obtained pollen were studied under constant laboratory conditions (20 ± 1°C, RH 65 ± 5%, photoperiod 16L: 8D h). Mites were kept individually at rearing units with ample quantity of almond (Prunus amygdalus Batch), apple (Malus domestica Borkh), apricot (Prunus armeniaca L.), cherry (Prunus avium L.), pear (Pyrus communis L.), plum (Prunus domestica L.) and walnut (Juglans regia L.) pollen as food source. Developmental time from egg to adult varied between the several pollen tested from 8.38 ± 0.08 to 9.58 ± 0.11 days for females and from 8.23 ± 0.12 and 9.07 ± 0.12 days for males. Female longevity varied from 11.53 ± 1.22 to 51.38 ± 2.45 days, while fecundity ranged from 22.84 ± 2.30 to 43.61 ± 3.78 eggs/female. The predator was unable to reproduce when feeding on walnut pollen. Data were submitted to life table analysis and values of the intrinsic rate of increase were derived, ranging from 0.079 to 0.146 (day⁻¹). The cumulative Weibull function that was used to describe the age specific survival of females produced excellent fits to the survival data. Results show that almond, plum, cherry and apricot pollen possess higher nutritional value for E. stipulatus than pear and apple pollen and thus may contribute in sustaining and increasing the predator population in field conditions. Walnut pollen can be utilized by the predator only to survive during short periods of time when principal or alternative food sources are scarce.