Shaohui Wang

Identification of jasmonic acid-associated microRNAs and characterization of the regulatory roles of the miR319/TCP4 module under root-knot nematode stress in tomato

Highlight Screening revealed that the action of miR319/TCP4 in serving as a systemic defensive responder and regulator that modulated the RKN systemic defensive response was mediated via JA.

Molecular cloning, characteristics and low temperature response of raffinose synthase gene in Cucumis sativus L.

Raffinose synthase (RS, EC2.4.1.82) is one of the key enzymes that channels sucrose into the raffinose family oligosaccharides (RFOs) biosynthetic pathway. However, the gene encoding RS is poorly characterized in cucumber (Cucumis sativus L.), which is a typical RFOs-translocating plant species. Here we isolated the gene encoding RS (CsRS) from the leaves of cucumber plants. The complete cDNA of CsRS consisted of 2552 nucleotides with an open reading frame encoding a polypeptide of 784 amino acid residues. Reverse transcription-polymerase chain reaction and RNA hybridization analysis revealed that expression of CsRS was the highest in leaves followed by roots, fruits, and stems. The RS activity was up-regulated and the raffinose content was high in the leaves of transgenic tobacco with over-expression of CsRS, while both the RS activity and the raffinose content decreased in the transgenic cucumber plants with anti-sense expression of CsRS. The expression of CsRS could be induced by low temperature and exogenous phytohormone abscisic acid (ABA). In cucumber growing under low temperature stress, CsRS expression, RS activity and raffinose content increased gradually in the leaves, the fruits, the stems and the roots. The most notable increase was observed in the leaves. Similarly, the expression of CsRS was induced in cucumber leaves and fruits with 200 μM and 150 μM ABA treatments, respectively.

Phloem unloading follows an extensive apoplasmic pathway in cucumber (Cucumis sativus L.) fruit from anthesis to marketable maturing stage

The phloem unloading pathway remains unclear in fruits of Cucurbitaceae, a classical stachyose-transporting species with bicollateral phloem. Using a combination of electron microscopy, transport of phloem-mobile symplasmic tracer carboxyfluorescein, assays of acid invertase and sucrose transporter, and [(14)C]sugar uptake, the phloem unloading pathway was studied in cucumber (Cucumis sativus) fruit from anthesis to the marketable maturing stage. Structural investigations showed that the sieve element-companion cell (SE-CC) complex of the vascular bundles feeding fruit flesh is apparently symplasmically restricted. Imaging of carboxyfluorescein unloading showed that the dye remained confined to the phloem strands of the vascular bundles in the whole fruit throughout the stages examined. A 37 kDa acid invertase was located predominantly in the cell walls of SE-CC complexes and parenchyma cells. Studies of [(14)C]sugar uptake suggested that energy-driven transporters may be functional in sugar trans-membrane transport within symplasmically restricted SE-CC complex, which was further confirmed by the existence of a functional plasma membrane sucrose transporter (CsSUT4) in cucumber fruit. These data provide a clear evidence for an apoplasmic phloem unloading pathway in cucumber fruit. A presumption that putative raffinose or stachyose transporters may be involved in soluble sugars unloading was discussed.

Low Light Stress Down-Regulated Rubisco Gene Expression and Photosynthetic Capacity During Cucumber (Cucumis sativus L.) Leaf Development

Low light stress is one of the most important factors affecting photosynthesis and growth in winter production of cucumber (Cucumis sativus L.) in solar greenhouses in northern China. Here, two genotypes of cucumber (Deltastar and Jinyan 2) are used to determine the effect of low light stress on Rubisco expression and photosynthesis of leaves from emergence to senescence. During leaf development, the net photosynthetic rate (PN), stomatal conductance (gs), Rubisco initial activity and activation state, transcript levels of rbcL and rbcS, and the abundance of rbcL and rbcS DNA in these two genotypes increase rapidly to reach maximum in 10-20 d, and then decrease gradually. Meanwhile, the actual photosystem II efficiency (?PSII) of cucumber leaves slowly increased in the early leaf developing stages, but it declined quickly in leaf senescent stages, accompanied by an increased non-photochemical quenching (NPQ). Moreover, PN, gs, initial Rubisco activity, and abundance of protein, mRNA and DNA of Rubisco subunits of leaves grown under 100 μmol m−2 s−1 are lower, and require more time to reach their maxima than those grown under 600 μmol m−2 s−1 during leaf development. All these results suggest that lower photosynthetic capacity of cucumber leaves from emergence to senescence under low light stress is probably due to down-regulated Rubisco gene expression in transcript and protein levels, and decreased initial and total activity as well as activation state of Rubisco. Deltastar performs better than Jinyan 2 under low light stress.

The Processes of Graft Union Formation in Tomato

Despite the long and widespread use of grafting in tomato cultivation, the structural changes that occur at the graft union during the early stages of the union remain poorly understood. In this study, the structural development of the graft union in tomato plants was examined 3, 5, 8, 11 and 14 days after grafting (DAG) using scanning electron microscopy (SEM) and a paraffin sectioning technique. The paraffin-sectioned images of the transection revealed that parenchymal cells from the graft union divided and proliferated in most combinations, and these cells could be detected at various stages of development. The SEM images of the scion and rootstock longitudinal sections showed that many interconnecting structures appeared at 8 DAG and were followed by a vascular rearrangement that did not connect the scion and rootstock. Vascular bundle bridges appeared at 11 DAG and connected the scion and rootstock. The connection of graft union between rootstock and scion is influenced by the plant growth condition. In our managed environment, period for formation of the connection structure formation between the rootstock and scion was 7-14 days after grafting.

Effects of exogenous abscisic acid pre-treatment of cucumber (Cucumis sativus) seeds on seedling growth and water-stress tolerance

Abstract The effect of treating cucumber seeds (Cucumis sativus ‘Zhongnong 12’) with exogenous abscisic acid (ABA) on seedling growth and various plant characteristics, including chlorophyll a fluorescence, net photosynthesis, antioxidant enzyme activity and water-stress tolerance under dehydration was investigated. The results showed that the photosynthesis rate (Pn), stomatal conductance (Gs), transpiration rate (Tr) and the apparent quantum yield of photosynthesis (AQY) all declined gradually, but the decline in ABA seed pre-treated seedlings (ASPS) was less than in non-ABA seed pre-treated seedlings (NASPS), especially for Pn during dehydration. Water use efficiency (WUE) was observed in ASPS, which was three times greater than that in NASPS, implying that the corresponding increase in WUE, calculated on the basis of water uptake, was the result of increased Pn and a reduction in transpiration in ASPS. During dehydration, the levels of electron transport rate (ETR), photochemical efficiency of PSII (Fv/Fm), photochemical quenching (qP), quantum efficiency of PSII photochemistry (ΦPSII) and the ratio of photochemicals (Pc) all decreased; in particular, the decline in NASPS was more than that in ASPS. The levels of non-photochemical quench (NPQ), ratio of antenna heat dissipation (Hd) and ratio of heat dissipation in PSII (Ex) all increased more in NASPS than in ASPS, suggesting that ABA could protect cucumber seedlings from the damage of photoinhibition by improved heat dissipation. Dehydration caused an increase in antioxidant enzyme activities in the first two days, especially in ASPS, and then declined slightly. In comparison with NASPS, ASPS showed a marked decrease in malondialdehyde (MDA) content during dehydration. On the other hand, rehydration resulted in nearly 100% recovery of Pn, Gs and Tr in ASPS, as well as chlorophyll fluorescence parameters, but only a small recovery in NASPS. These results suggest that imbibition of cucumber seeds with ABA protected cucumber seedlings from water-loss damage and increased their resistance or tolerance of dehydration, especially improving their WUE by improved Pn rather than by reduced water uptake under dehydration. The beneficial effect of exogenous ABA applications on reducing susceptibility to gradual dehydration of cucumber seedlings was confirmed in our study by applying ABA to imbibition cucumber seeds.

Effects of exogenous abscisic acid on leaf carbohydrate metabolism during cucumber seedling dehydration

Cucumber (Cucumis sativus L.) seeds were pretreated with exogenous abscisic acid (ABA) prior to germination. After germination, seedlings with three leaves were exposed to gradual dehydration. The effects of ABA on photosynthetic rate (Pn), daily water loss (WL) and water utilization efficiency (WUE) during dehydration were investigated, in addition to the variation of carbohydrates in leaves. ABA improved the Pn, WL and WUE of cucumber seedlings during dehydration. After rehydration, the seedlings pretreated with ABA showed a higher recovery in Pn, WL and WUE, as compared to those without an ABA pretreatment. Subsequent to dehydration, concentration of stachyose, raffinose, sucrose, glucose, and fructose increased in seedlings pretreated with ABA. Dehydration altered the proportions of the sugars in the total carbohydrates, and accelerated the accumulation of stachyose, raffinose and sucrose. After rehydration, carbohydrate concentrations of seedlings pretreated with ABA recovered to levels observed prior to dehydration. These results demonstrated that pretreatment of seeds with exogenous ABA enhanced carbohydrate tolerance to dehydration of cucumber seedlings.

The use of toluidine blue staining combined with paraffin sectioning and the optimization of freeze-thaw counting methods for analysing root-knot nematodes in tomato

Root-knot nematodes (RKNs) (Meloidogyne spp.) are one of the most damaging crop pests and cause severe yield losses worldwide. Easier and more efficient methods are needed for assessing nematode infestations and host plant resistance. In the present study, we combined toluidine blue staining and paraffin sectioning to observe the establishment of giant cells (GCs; redifferentiated cells induced by nematode feeding) and the developmental status of RKNs in tomato (Solanum lycopersicum). The number of RKNs was counted using a modified freeze-thaw counting method. Finally, three tomato lines with different levels of RKN resistance, but otherwise identical genetic backgrounds, were tested to verify the applicability of the combined method. The results suggested that using toluidine blue staining combined with paraffin sectioning and freeze-thaw counting accurately reflects the level of RKN resistance of tomato plants.

Protective enzymes and genes related to the JA pathway are involved in the response to root-knot nematodes at high soil temperatures in tomatoes carrying Mi-1

Root-knot nematodes (RKNs; Meloidogyne spp.) are obligate endoparasites that infect a large number of crop plants and cause severe yield losses. Tomato cultivars carrying the Mi-1 gene conferring root-knot nematode resistance have been widely used, but this gene loses its effectiveness at soil temperatures above 28°C. In this study, the mechanism of the loss of resistance to RKNs at high soil temperatures was examined using LA0655 (Solanum lycopersicum cv. Anahu), which contains the Mi-1 gene. It was found that high soil temperatures delayed the expression of the Mi-1 gene and reduced the activities of superoxide dismutase, peroxidase, chitinase, and β-1,3-glucanase. Although genes in the jasmonic acid (JA) pathway exhibited an obvious response at high soil temperatures, this response could not prevent the invasion of RKNs; indeed, at 30 days after inoculation with RKNs, the plants produced large numbers of root knots and egg masses at a soil temperature of 32°C.

Comparison of manual and automatic processing of biological samples for electron microscopy

The ultrastructure of a sample can be observed by electron microscopy (EM), which has become an indispensable research tool in morphological studies. However, EM sample preparation techniques are complicated and time‐consuming, with a high labor cost. The current study was conducted to compare the conventional manual and automated methods for sample processing and post‐staining for electron microscopy. Automated sample processing reduces OsO4 contamination, improves the efficiency of sample preparation and is easy to use. Therefore, the results of their study provide a practical and feasible method for the preparation of biological samples for electron microscopy.