G protein and PLDδ are involved in JA to regulate osmotic stress responses in Arabidopsis thaliana

Jasmonic acid (JA) is regarded as an endogenous regulator which plays an important role in regulating plant growth, development and stress response. Using the seedlings of A. thaliana ecotype Col-0 (wild-type, WT), phospholipase Dδ (PLDδ) deficient mutant (pldδ), the G protein α subunit (GPA1) deficient mutant (gpa1-4), 9-Lipoxygenase (9-LOX) deficient mutants (lox1 and lox5) as materials, the effects of JA responding to osmotic stress and the functions of G protein and PLDδ in this response were investigated. The results showed that GPA1 involved in the regulation of JA to PLDδ under osmotic stress. Both GPA1 and PLDδ participated in the regulation of JA on the seed germination and osmotic tolerance. Exogenous MeJA reduced the EL and MDA in WT, but increased the EL and MDA in gpa1-4 and pldδ, indicating that GPA1 and PLDδ were involved in the protection of JA on the membrane. The genes expression levels, and the activities of PLDδ and LOX1 were significantly induced by osmotic stress. The LOX activity and JA content in pldδ seedings were lower obviously than those in WT, but were markedly increased and were higher than WT after applying phosphatidic acid (PA). These results demonstrated that JA responded to osmotic stress by regulating G protein and PLDδ in A. thaliana. PLDδ was located upstream of 9-LOX and involved in the JA biosynthesis.     

Phospholipase Dδ negatively regulates plant thermotolerance by destabilizing cortical microtubules in Arabidopsis

The pattern of cortical microtubule arrays plays an important role in plant growth and adaptation in response to hormonal and environmental changes. Cortical microtubules are connected with the plasma membrane (PM); however, how the membrane affects cortical microtubule organization is not well understood. Here, we showed that phospholipase Dδ (PLDδ) was associated with the PM and co‐localized with microtubules in cells. In vitro analysis revealed that PLDδ bound to microtubules, resulting in microtubule disorganization. Site‐specific mutations that decreased PLDδ enzymatic activity impaired its effects on destabilizing microtubule organization. Heat shock transiently activated PLDδ, without any change of its PM localization, triggering microtubule dissociation from PM and depolymerization and seedling death in Arabidopsis, but these effects were alleviated in pldδ knockout mutants. Complementation of pldδ with wild‐type PLDδ, but not mutated PLDδ, restored the phenotypes of microtubules and seedling survival to those of wild‐type Arabidopsis. Thus, we conclude that the PM‐associated PLDδ negatively regulates plant thermotolerance via destabilizing cortical microtubules, in an activity‐dependent manner, rather than its subcellular translocation.     

Phospholipase Dδ is involved in nitric oxide-induced stomatal closure

Nitric oxide (NO) has recently emerged as a second messenger involved in the complex network of signaling events that regulate stomatal closure. Little is known about the signaling events occurring downstream of NO. Previously, we demonstrated the involvement of phospholipase D (PLD) in NO signaling during stomatal closure. PLDδ, one of the 12 Arabidopsis PLDs, is involved in dehydration stress responses. To investigate the role of PLDδ in NO signaling in guard cells, we analyzed guard cells responses using Arabidopsis wild type and two independent pldδ single mutants. In this work, we show that pldδ mutants failed to close the stomata in response to NO. Treatments with phosphatidic acid, the product of PLD activity, induced stomatal closure in pldδ mutants. Abscisic acid (ABA) signaling in guard cells involved H₂O₂ and NO production, both required for ABA-induced stomatal closure. pldδ guard cells produced similar NO and H₂O₂ levels as the wild type in response to ABA. However, ABA- or H₂O₂-induced stomatal closure was impaired in pldδ plants. These data indicate that PLDδ is downstream of NO and H₂O₂ in ABA-induced stomatal closure.     

Phospholipases Dα and δ are involved in local and systemic wound responses of cotton (G. hirsutum)

Phospholipases D (PLDs) catabolize structural phospholipids to produce phosphatidic acid (PtdOH), a lipid playing central role in signalling pathways in animal, yeast and plant cells. In animal cells two PLD genes have been studied while in model plant Arabidopsis twelve genes exist, classified in six classes (α-ζ). This underlines the role of these enzymes in plant responses to environmental stresses. However, information concerning the PLD involvement in the widely cultivated and economically important cotton plant responses is very limited. The aim of this report was to study the activity of conventional cotton PLD and its participation in plant responses to mechanical wounding, which resembles both biotic and abiotic stresses. PLDα activity was identified and further characterized by transphosphatidylation reaction. Upon wounding, cotton leaf responses consist of an acute in vitro increase of PLDα activity in both wounded and systemic tissue. However, determination of the in vivo PtdOH levels under the same wounding conditions revealed a rapid PtdOH formation only in wounded leaves and a late response of a PtdOH increase in both tissues. Εxpression analysis of PLDα and PLDδ isoforms showed mRNA accumulation of both isoforms in the wounded tissue, but only PLDδ exerts a high and sustainable expression in systemic leaves, indicating that this isoform is mainly responsible for the systemic wound-induced PtdOH production. Therefore, our data suggest that PLDα and PLDδ isoforms are involved in different steps in cotton wound signalling.     

Banana Ovate Family Protein MaOFP1 and MADS-Box Protein MuMADS1 Antagonistically Regulated Banana Fruit Ripening

The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening.     

Intergeneric Hybridisation between Litchi (Litchi chinensis Sonn.) and Longan (Dimocarpus longan Lour.)

The breeding barriers between commercial litchi (Litchi chinensisSonn.) and longan (Dimocarpus longan Lour.) cultivars were investigatedby conducting reciprocal pollinations. This work has shown thatit is possible to generate intergeneric hybrids using litchias the female parent. Investigation of comparative in vivo pollentube growth demonstrated that there is discrimination againstcross- compared to self-pollen at all sites in the pistil. Pollentubes were frequently observed in the ovary after cross-pollinationin litchi but rarely in longan. Fruit production was reducedafter crossing in both longan and litchi. Isozyme analysis usingphosphoglucose isomerase revealed that hybrid progeny only developedwhen litchi was the maternal parent. Morphologically the hybridplants were similar to the maternal parent but leaves were smaller.Three types of seeds developed in litchi following pollinationwith longan pollen. These were (1) normal seeds with a developedtesta and embryo, (2) seeds with aborted embryos but normaltesta development, and (3) seedless fruit where the ovule remainedthe same size as at anthesis without further development ofembryo or testa. The potential germplasm available to improvethese crops within the Sapindaceae is discussed.Copyright 1994,1999 Academic Press Litchi, Litchi Longan, Dimocarpus, hybridisation, isozyme     

Functional analysis of phospholipase Dδ family in tobacco pollen tubes

Phosphatidic acid (PA), an important signalling and metabolic phospholipid, is predominantly localized in the subapical plasma membrane (PM) of growing pollen tubes. PA can be produced from structural phospholipids by phospholipase D (PLD), but the isoforms responsible for production of PM PA were not identified yet and their functional roles remain unknown. Following genome‐wide bioinformatic analysis of the PLD family in tobacco, we focused on the pollen‐overrepresented PLDδ class. Combining live‐cell imaging, gene overexpression, lipid‐binding and structural bioinformatics, we characterized five NtPLDδ isoforms. Distinct PLDδ isoforms preferentially localize to the cytoplasm or subapical PM. Using fluorescence recovery after photobleaching, domain deletion and swapping analyses we show that membrane‐bound PLDδs are tightly bound to PM, primarily via the central catalytic domain. Overexpression analyses suggested isoform PLDδ3 as the most important member of the PLDδ subfamily active in pollen tubes. Moreover, only PLDδ3 shows significant constitutive PLD activity in vivo and, in turn, PA promotes binding of PLDδ3 to the PM. This forms a positive feedback loop leading to PA accumulation and the formation of massive PM invaginations. Tightly controlled production of PA generated by PLDδ3 at the PM is important for maintaining the balance between various membrane trafficking processes that are crucial for plant cell tip growth.     

Developmental regulation of phospholipase D in tomato fruits

The catabolism of phospholipids initiated by phospholipase D (PLD, EC 3.1.4.4) is an inherent feature of developmental processes that include fruit growth and ripening. In cherry tomatoes (Lycopersicon esculentum Mill.), soluble and membrane-associated PLD activities increased during fruit development, which peaked at the mature green and orange stages. The increase in PLD activity was associated with a similar increase in the intensity of a 92 kDa band as demonstrated by western blot analysis. A full-length cDNA having 2430 bp and encoding a putative polypeptide with 809 amino acids, was isolated using tomato RNA, RT-PCR and 5' and 3' rapid amplification of cloned ends (RACE). Analysis of the primary and secondary structures showed the presence of the C2 domain, the PLD domain and several other features characteristic of PLD alpha. Microtom tomato plants transformed with antisense PLD alpha cDNA, were similar to untransformed plants and showed normal fruit set and development. The ethylene climacteric was delayed by over 7 d in the antisense PLD fruits, indicative of a slower ripening process. The leaves and unripened fruits of antisense PLD microtom plants possessed lowered PLD activity and PLD protein, as demonstrated by western blotting. However, during ripening, PLD activity in the transgenic fruits was maintained at a higher level than that in the untransformed control. Immunolocalization of PLD in microtom tomato fruits revealed the cytosol-membrane translocation of PLD during fruit development. The ripe fruits of antisense PLD celebrity plants possessed lowered PLD expression and activity and showed increased firmness and red colour. These results suggest that the expression of antisense PLD cDNA could be variable in different tomato varieties. The potential role of PLD in ethylene signal transduction events is discussed.     

药剂输液滴干对龙眼成花的影响

虹吸输液滴干技术作为一种新型的输液技术,能显著提高龙眼对调节剂的利用率。该研究利用虹吸输液滴干技术来研究氯酸钾与多效唑对龙眼成花的影响,同时探索从花芽生理分化期开始控制龙眼冲梢的方法。结果表明：氯酸钾输液滴干不但能够促使龙眼提早开花,而且能够提高其成花率、抽穗率、雌雄花比例与坐果率。不同品种龙眼对氯酸钾的敏感度不同,‘石硖’对氯酸钾最为敏感,但稳定性低;‘储良’对氯酸钾的敏感性低,但稳定度高;‘桂香’对氯酸钾的敏感性与稳定度居中。从整体上看,‘石硖’的处理效果最好,最佳处理次数为3次,每次施用7.5 L浓度为1 g·L-1的氯酸钾,处理后枝梢成花率、枝梢抽穗率、雌雄花比例、坐果率分别为53.67％、59.38％、16.01和19.58％。多效唑输液滴干对‘桂龙1号’的控梢效果显著,有效促进了龙眼成花,并以160 mg·L-1的多效唑溶液处理效果最佳,抽梢率为15.16％,抽穗率、雌花率、花穗长、花穗宽和坐果率分别为96.53％、10.36％、21.95 cm、12.96 cm和38.37％。药剂输液滴干技术施用氯酸钾和多效唑对于龙眼成花坐果有积极作用,该研究结果为实现高效、省工、环保的果园管理模式提供了借鉴。     

A novel aspartic acid protease gene from pineapple fruit (Ananas comosus): Cloning,characterization and relation to postharvest chilling stress resistance

A full-length cDNA encoding a putative aspartic acid protease (AcAP1) was isolated for the first time from the flesh of pineapple (Ananas comosus) fruit. The deduced sequence of AcAP1 showed all the common features of a typical plant aspartic protease phytepsin precursor. Analysis of AcAP1 gene expression under postharvest chilling treatment in two pineapple varieties differing in their resistance to blackheart development revealed opposite trends. The resistant variety showed an up-regulation of AcAP1 precursor gene expression whereas the susceptible showed a down-regulation in response to postharvest chilling treatment. The same trend was observed regarding specific AP enzyme activity in both varieties. Taken together our results support the involvement of AcAP1 in postharvest chilling stress resistance in pineapple fruits.     

磷脂酶Dδ参与植物的低温驯化过程

对经低温驯化和未经低温驯化的磷脂酶Dδ（PLDδ）基因敲除突变体与野生型植株进行冻害胁迫处理后,比较2种基因型植株的抗冻性。结果发现,经低温驯化的PLDδ敲除突变体的抗冻性明显低于野生型,而未经低温驯化的PLD礅除突变体与野生型的抗冻性没有显著差异,表明PLDδ参与植物的低温驯化过程。对PLDδ的作用途径进行分析,发现PLDδ在低温驯化过程中不参与抗氧化酶活性的调节,对脯氨酸和可溶性糖的积累起负调节作用,但是参与低温信号转导物质ABA诱导抗冻性的过程。     

Cytosolic glyceraldehyde-3-phosphate dehydrogenases interact with phospholipase Dδ to transduce hydrogen peroxide signals in the Arabidopsis response to stress

Reactive oxygen species (ROS) are produced in plants under various stress conditions and serve as important mediators in plant responses to stresses. Here, we show that the cytosolic glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenases (GAPCs) interact with the plasma membrane-associated phospholipase D (PLDδ) to transduce the ROS hydrogen peroxide (H(2)O(2)) signal in Arabidopsis thaliana. Genetic ablation of PLDδ impeded stomatal response to abscisic acid (ABA) and H(2)O(2), placing PLDδ downstream of H(2)O(2) in mediating ABA-induced stomatal closure. To determine the molecular link between H(2)O(2) and PLDδ, GAPC1 and GAPC2 were identified to bind to PLDδ, and the interaction was demonstrated by coprecipitation using proteins expressed in Escherichia coli and yeast, surface plasmon resonance, and bimolecular fluorescence complementation. H(2)O(2) promoted the GAPC-PLDδ interaction and PLDδ activity. Knockout of GAPCs decreased ABA- and H(2)O(2)-induced activation of PLD and stomatal sensitivity to ABA. The loss of GAPCs or PLDδ rendered plants less responsive to water deficits than the wild type. The results indicate that the H(2)O(2)-promoted interaction of GAPC and PLDδ may provide a direct connection between membrane lipid-based signaling, energy metabolism and growth control in the plant response to ROS and water stress.     

Cloning and Expression Analysis of Litchi (Litchi Chinensis Sonn.) Polyphenol Oxidase Gene and Relationship with Postharvest Pericarp Browning

Polyphenol oxidase (PPO) plays a key role in the postharvest pericarp browning of litchi fruit, but its underlying mechanism remains unclear. In this study, we cloned the litchi PPO gene (LcPPO, {"type":"entrez-nucleotide","attrs":{"text":"JF926153","term_id":"350601663","term_text":"JF926153"}}JF926153), and described its expression patterns. The LcPPO cDNA sequence was 2120 bps in length with an open reading frame (ORF) of 1800 bps. The ORF encoded a polypeptide with 599 amino acid residues, sharing high similarities with other plant PPO. The DNA sequence of the ORF contained a 215-bp intron. After carrying out quantitative RT-PCR, we proved that the LcPPO expression was tissue-specific, exhibiting the highest level in the flower and leaf. In the pericarp of newly-harvested litchi fruits, the LcPPO expression level was relatively high compared with developing fruits. Regardless of the litchi cultivar and treatment conditions, the LcPPO expression level and the PPO activity in pericarp of postharvest fruits exhibited the similar variations. When the fruits were stored at room temperature without packaging, all the pericarp browning index, PPO activity and the LcPPO expression level of litchi pericarps were reaching the highest in Nandaowuhe (the most rapid browning cultivar), but the lowest in Ziniangxi (the slowest browning cultivar) within 2 d postharvest. Preserving the fruits of Feizixiao in 0.2-μm plastic bag at room temperature would decrease the rate of pericarp water loss, delay the pericarp browning, and also cause the reduction of the pericarp PPO activity and LcPPO expression level within 3 d postharvest. In addition, postharvest storage of Feizixiao fruit stored at 4°C delayed the pericarp browning while decreasing the pericarp PPO activity and LcPPO expression level within 2 d after harvest. Thus, we concluded that the up-regulation of LcPPO expression in pericarp at early stage of postharvest storage likely enhanced the PPO activity and further accelerated the postharvest pericarp browning of litchi fruit.     

PPARδ is pro-tumorigenic in a mouse model of COX-2-induced mammary cancer

Cyclooxygenase-2 (COX-2), overexpressed in inflammatory conditions and cancer, regulates angiogenesis and tumorigenesis via the production of biologically active prostanoids. Previously, we showed that COX-2 over-expression in the mammary gland of transgenic mice induces an angiogenic switch and transforms the mammary epithelium into invasive mammary carcinoma. Since COX-2-derived prostanoids can activate the nuclear receptor PPARδ, we crossed Pparδ^?/? mice with COX-2 transgenic mice in the FVB/N background. PPARδ was expressed constitutively in the mammary gland of virgin, pregnant and lactating mice. Mammary hyperplasia and tumorigenesis in the COX-2 transgenic mice was markedly reduced in the Pparδ^?/? mice compared to their wild type counterparts. Analysis of the mammary tissues indicated that immunoreactive Ki-67, cyclin D1 and phosphorylated histone 3 (Phospho H3) were reduced in Pparδ^?/? mice, suggesting that PPARδ activation regulates cell proliferation in the mammary gland. We postulate that activation of the nuclear receptor PPARδ by COX-2-derived prostanoids may be involved in the proliferation of mammary epithelial cells and therefore contribute to mammary cancer development.     

Impact of postharvest exogenous γ-aminobutyric acid treatment on cucumber fruit in response to chilling tolerance

Low-temperature storage is generally used to extend postharvest lifetime and to inhibit decay of cucumber fruit, but it also enhances the intensity of chilling injury. The capability of γ-aminobutyric acid to enhance antioxidant enzyme activities and reduce chilling injury was studied in cucumber (Cucumis sativus L.) fruit stored at 1 °C for 5 weeks. The purpose of this study was to define if the GABA-induced modification in antioxidant system and phospholipase activity is linked to the reduced chilling injury in cold-stored cucumber fruit. Alleviation of chilling injury by GABA treatment was related to increased content of proline, endogenous GABA and enhanced activities of CAT and SOD, together with reduced activities of PLC, PLD and LOX. We suggest that PLC, LOX and PLD are associated with chilling injury initiation by involvement in a signaling pathway and membrane deterioration. Therefore the results obtained in this study suggest GABA’s potential for postharvest applications for reducing chilling injury symptom in cucumber fruit.     

Comparative Analysis Reveals Dynamic Changes in miRNAs and Their Targets and Expression during Somatic Embryogenesis in Longan (Dimocarpus longan Lour.)

Somatic embryogenesis (SE), which resembles zygotic embryogenesis, is an essential component of the process of plant cell differentiation and embryo development. Although microRNAs (miRNAs) are important regulators of many plant develop- mental processes, their roles in SE have not been thoroughly investigated. In this study, we used deep-sequencing, computational, and qPCR methods to identify, profile, and describe conserved and novel miRNAs involved in longan (Dimocarpus longan) SE. A total of 643 conserved and 29 novel miRNAs (including star strands) from more than 169 miRNA families were identified in longan embryogenic tissue using Solexa sequencing. By combining computational and degradome sequencing approaches, we were able to predict 2063 targets of 272 miRNAs and verify 862 targets of 181 miRNAs. Target annotation revealed that the putative targets were involved in a broad variety of biological processes, including plant metabolism, signal transduction, and stimulus response. Analysis of stage- and tissue-specific expressions of 20 conserved and 4 novel miRNAs indicated their possible roles in longan SE. These miRNAs were dlo-miR156 family members and dlo-miR166c* associated with early embryonic culture developmental stages; dlo-miR26, dlo-miR160a, and families dlo-miR159, dlo-miR390, and dlo-miR398b related to heart-shaped and torpedo- shaped embryo formation; dlo-miR4a, dlo-miR24, dlo-miR167a, dlo-miR168a*, dlo-miR397a, dlo-miR398b.1, dlo-miR398b.2, dlo-miR808 and dlo-miR5077 involved in cotyledonary embryonic development; and dlo-miR17 and dlo-miR2089*-1 that have regulatory roles during longan SE. In addition, dlo-miR167a, dlo-miR808, and dlo-miR5077 may be required for mature embryo formation. This study is the first reported investigation of longan SE involving large-scale cloning, characterization, and expression profiling of miRNAs and their targets. The reported results contribute to our knowledge of somatic embryo miRNAs and provide insights into miRNA biogenesis and expression in plant somatic embryo development.