Transgenic zoysiagrass (Zoysia japonica) plants obtained by Agrobacterium-mediated transformation

Zoysiagrass (Zoysia japonica Steud.) is an important turfgrass that spreads by stolons and rhizomes. By exploring the potential of direct shoot formation from stolons, we developed a straightforward and efficient transformation protocol without callus induction and propagation. Sterilized stolon nodes were infected and co-cultivated with Agrobacterium tumefaciens harboring pCAMBIA vectors. Hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin was used as the selection agent. Both green and albino shoots were directly regenerated from the infected stolon nodes 4–5 weeks after hygromycin selection. Greenhouse-grown plants were obtained 10–12 weeks after Agrobacterium-mediated transformation. Based on the number of transgenic plants obtained and the number of stolon nodes infected, a transformation frequency of 6.8% was achieved. Stable integration of the transgenes in the plant genome was demonstrated by PCR and Southern blot hybridization analyses. Expression of the transgenes was confirmed by RT-PCR analysis and GUS staining. The new transformation system opens up new opportunities for the functional characterization of genes and promoters and the development of novel germplasm in zoysiagrass.     

Gene expression involved in dark-induced leaf senescence in zoysiagrass (Zoysia japonica)

Zoysiagrass (Zoysia japonica) is one of the important turfgrass species. Extending green period of zoysiagrass via delaying leaf senescence will make this species have more potential in the turfgrass industry. In this study, we found that zoysiagrass seedlings treated with GA₃ could delay the leaf senescence induced by darkness. To study expression of genes responsive to staying green in zoysiagrass, suppression subtractive hybridization (SSH) was used to identify differentially expressed genes between non-GA₃-treated and GA₃-treated seedlings subjected to darkness. A total of 307 ESTs were generated, of which 226 ESTs clustered into 54 contigs and 81 were singlets. Differentially expressed genes selected by subtractions were classified into six categories according to their putative functions generated by BLAST analysis. Expression of five selected genes, Met, SAM, V-ATPase, Cry (Cryptochrome gene), and An (diphthine synthase gene) were examined by RT-PCR and Real-time PCR. Both RT-PCR and Real-time PCR results demonstrated that the differential expressions of these genes were attributable to delaying senescence by exogenously applied gibberellic acid. This is the first genome-wide study of senescence in a species of turfgrass.     

Effect of salicylic acid pretreatment on cadmium toxicity in wheat

Cadmium (100, 400 and 1000 μM CdCl₂) treatments resulted in the inhibition of root dry biomass, root elongation and increased Cd accumulation in wheat (Triticum aestivum L.) roots. Further, these treatments decreased relative water content, chlorophyll content, ¹⁴CO₂-fixation, activities of phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase and abscisic acid content while increased malondialdehyde, hydrogen peroxide and free proline contents. Chloroplast and root ultrastructure was also changed. Pretreatment of seeds with SA (500 μM) for 20 h resulted in amelioration of these effects.     

Treatment with acetyl salicylic acid protects muskmelon seedlings against drought stress

Salicylic acid (SA) is a common plant-produced signal molecule that is responsible for inducing tolerance to a number of biotic and abiotic stresses. An experiment was, therefore, conducted to test whether acetyl salicylic acid (ASA) application at various concentrations through seed soaking or foliar spray would protect muskmelon [Cucumis melo L. (reticulatus group)] seedlings, subjected to drought stress. Twenty-three-day-old plants pre-treated with ASA (0, 0.1, 0.25, 0.50 or 1.0 mM) were subjected to drought stress for 1 week in a greenhouse. ASA applied either through seed soaking or through foliar spray was effective within the range of 0.1–1 mM in providing drought stress protection in muskmelon seedlings; however, there was no difference between application methods indicating that both methods provided similar levels of protection. ASA significantly affected all seedling growth and stress indicator variables measured except leaf number and root dry weight. The best protection appeared to be obtained from seedlings pre-treated with lower concentrations of ASA. Even though both methods provided similar means of protection, due to its simplicity and practicality, soaking muskmelon seeds prior to sowing in up to 0.5 mM ASA would be a more desirable method.     

Characterization of 30 new microsatellite markers,developed from enriched genomic DNA library of zoysiagrass Zoysia japonica Steud.

The present study reports the development of 30 polymorphic microsatellite markers for zoysiagrass Zoysia japonica Steud. The 30 markers produced a total of 125 alleles with an average of 4.2 alleles per locus. Values for observed and expected heterozygosities ranged from 0.10 to 0.95 and from 0.15 to 0.81, respectively. At significance threshold (P < 0.05), 11 loci deviated from Hardy–Weinberg equilibrium, whereas significant linkage disequilibrium values were observed between 16 pairs of loci. These markers may provide information needed to select genetically diverse parents for developing breeding and mapping populations of zoysiagrass.     

Acetyl salicylic acid (Aspirin) and salicylic acid induce multiple stress tolerance in bean and tomato plants

The hypothesis that physiologically activeconcentrations of salicylic acid (SA) and itsderivatives can confer stress tolerance in plants wasevaluated using bean (Phaseolus vulgaris L.) andtomato (Lycopersicon esculentum L.). Plantsgrown from seeds imbibed in aqueous solutions (0.1--0.5 mM) of salicylic acid or acetyl salicylic acid(ASA) displayed enhanced tolerance to heat, chillingand drought stresses. Seedlings acquired similarstress tolerance when SA or ASA treatments wereapplied as soil drenches. The fact that seedimbibition with SA or ASA confers stress tolerance inplants is more consistent with a signaling role ofthese molecules, leading to the expression oftolerance rather than a direct effect. Induction ofmultiple stress tolerance in plants by exogenousapplication of SA and its derivatives may have asignificant practical application in agriculture,horticulture and forestry.     

水杨酸对黄瓜幼苗抗高温胁迫能力的影响

对津绿2号黄瓜四叶期的幼苗分别喷施0、50、100和200μmol·L-1的水杨酸(SA)溶液,24h后以42℃的高温胁迫24h。结果表明:不同浓度的SA溶液均可降低黄瓜叶片中的相对电导率和丙二醛(MDA)含量,增加SOD活性,以50μmol·L-1的效果最优。但相对电导率和MDA含量的降幅及SOD活性的增幅则随SA浓度的增加而减小。     

Exogenous 4-hydroxybenzoic acid and salicylic acid modulate the effect of short-term drought and freezing stress on wheat plants

Exogenous salicylic acid has been shown to confer tolerance against biotic and abiotic stresses. In the present work the ability of its analogue, 4-hydroxybenzoic acid to increase abiotic stress tolerance was demonstrated: it improved the drought tolerance of the winter wheat (Triticum aestivum L.) cv. Cheyenne and the freezing tolerance of the spring wheat cv. Chinese Spring. Salicylic acid, however, reduced the freezing tolerance of Cheyenne and the drought tolerance of Chinese Spring, in spite of an increase in the guaiacol peroxidase and ascorbate peroxidase activity. The induction of cross tolerance between drought and freezing stress was observed: drought acclimation increased the freezing tolerance of Cheyenne plants and cold acclimation enhanced the drought tolerance. The induction of drought tolerance in Cheyenne was correlated with an increase in catalase activity.     

Isolation, characterization and mapping of simple sequence repeat markers in zoysiagrass (Zoysia spp.)

The genus Zoysia consists of 16 species that are naturally distributed on sea coasts and grasslands around the Pacific. Of these, Zoysia japonica, Zoysia matrella, and Zoysia tenuifolia are grown extensively as turfgrasses, and Z. japonica is also used as forage grass in Japan and other countries in East Asia. To develop simple sequence repeat (SSR) markers for zoysiagrass (Zoysia spp.), we used four SSR-enriched genomic libraries to isolate 1,163 unique SSR clones. All four libraries contained a high percentage of perfect clones, ranging from 67.1 to 96.0%, and compound clones occurred with higher frequencies in libraries A (28.6%) and D (11.6%). From these clones, we developed 1,044 SSR markers when we tested all 1,163 SSR primer pairs. Using all 1,044 SSR markers, we tested one screening panel consisting of eight Zoysia clones for testing PCR amplifications, from which five unrelated clones, among the eight, were used for polymorphism assessment, and found that the polymorphic information content ranged from 0 (monomorphic loci) to 0.88. Of the 1,044 SSR markers, 170 were segregated in our mapping population and we mapped 161 on existing amplified fragment length polymorphism-based linkage groups, using this mapping population. These SSR markers will provide an ideal marker system to assist with gene targeting, quantitative trait locus mapping, variety or species identification, and marker-assisted selection in Zoysia species.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Somatic embryogenesis and protoplast culture in Japanese lawngrass (Zoysia japonica)

Embryogenic callus cultures were initiated from the mature caryopses ofZoysia japonica. Plant regeneration was through precocious germination of somatic embryos. Protoplasts were isolated from the callus and cultured in a medium solidified with agarose. Numerous calli were recovered after transferring protocolonies onto an agar medium.Abbreviations MS Murashige & Skoog - CH Casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-Morpholino)ethanesulfoic acid     

氮肥对受践踏胁迫结缕草草坪恢复生长的影响

以日本结缕草草坪为试材,采用田间试验,研究在不同践踏胁迫下,施用氮肥对草坪生长的影响。结果表明,施氮肥有利于受践踏草坪的恢复生长,在不同程度践踏胁迫下,施氮素8 g/m2,可不同程度地增加草坪的密度、株高生长量和地上生物量;总叶绿素含量比对照明显增加。     

外源水杨酸对干旱胁迫下小冠花种子萌发及幼芽生理特性的影响

以豆科牧草“绿宝石”小冠花为试材,研究PEG-6000(浓度8%和12%)模拟干旱胁迫下不同浓度外源水杨酸(0、0.5、1.0和2.0 mmol·L^-1)对小冠花种子萌发和幼芽生理特性的影响.结果表明: 0.5～1.0 mmol·L^-1水杨酸显著提高了干旱胁迫下小冠花种子发芽率、发芽势、发芽指数、活力指数和芽长,12%PEG胁迫下1.0 mmol·L^-1水杨酸处理小冠花幼芽干质量显著高于干旱处理.0.5～1.0 mmol·L^-1水杨酸处理显著提高了干旱胁迫下小冠花幼芽脯氨酸、可溶性蛋白含量,显著提高了过氧化氢酶、过氧化物酶、超氧化物歧化酶活性,显著降低了幼芽细胞电解质渗透率、H₂O₂含量、O₂^-·产生速率,其中以1.0 mmol·L^-1水杨酸处理效果最好.水杨酸浓度超过2.0 mmol·L^-1时对干旱胁迫没有缓解效应.表明适宜浓度的水杨酸(0.5～1.0 mmol·L^-1)可以提高小冠花幼芽渗透调节能力和抗氧化能力,促进小冠花生长,缓解干旱胁迫伤害.     

NaCl与干旱双重胁迫下黑果枸杞幼苗对外源水杨酸的生理响应

干旱、盐分已成为限制植物生长发育的主要因子,在干旱与NaCl双重胁迫下植物的生长发育受到一定影响。为了探究黑果枸杞（Lycium ruthenicum）对盐旱逆境的适应性,该文采用盆栽试验,研究NaCl与干旱胁迫共同作用对其幼苗生长的影响,并观察盐旱逆境下黑果枸杞幼苗对外源水杨酸（SA）的生理响应,探究提高NaCl与干旱胁迫下黑果枸杞幼苗的存活率。结果表明：外源SA(0.1、0.5 mmol·L^-1)处理下,盐旱双重胁迫下黑果枸杞叶内可溶性糖、可溶性蛋白和脯氨酸含量有所增加,而丙二醛（MDA）含量显著降低（P<0.05）,过氧化氢酶（CAT）、过氧化物酶（POD）和超氧化物歧化酶（SOD）活性上升,且0.5 mmol·L^-1 SA处理效果优于0.1 mmol·L^-1 处理。综上结果可知,黑果枸杞对于轻度盐旱胁迫具有一定的适应能力,适宜浓度SA可提高盐旱逆境中黑果枸杞叶内渗透调节物质含量及抗氧化酶活性,该研究为进一步了解盐旱双重胁迫下黑果枸杞幼苗的生长发育提供相关理论依据。     

干旱胁迫下外源水杨酸对黄瓜幼苗膜脂过氧化和光合特性的影响

为了探讨外源水杨酸(SA)提高植物抗旱性的相关机制,研究了干旱胁迫下(基质含水量为饱和持水量的60％和50％),根际施用外源SA对黄瓜幼苗生长、膜脂过氧化、脯氨酸积累、水分利用效率、净光合速率(Pn)和叶绿素荧光参数的影响.结果表明:SA处理能够缓解干旱胁迫对黄瓜幼苗生长、Pn和水分利用效率的抑制,减小膜脂过氧化程度,促进脯氨酸的积累;添加外源SA显著减小了干旱胁迫下黄瓜幼苗的PSⅡ最大光化学效率、PSⅡ实际光化学效率、PSⅡ潜在活性、PSⅡ有效光化学效率和光化学猝灭系数的下降幅度,抑制了非光化学猝灭系数的升高.添加外源SA可以缓解干旱胁迫造成的膜脂过氧化对膜系统的氧化损伤,并通过增强PSⅡ反应中心活性提高了Pn,有助于水分的利用,同时增大渗透调节能力,减少水分的散失,提高水分利用效率,从而增强植株对干旱的适应能力.     

Cross-talk between gibberellins and salicylic acid in early stress responses in Arabidopsis thaliana seeds

Salicylic acid (SA) is a plant hormone mainly associated with the induction of defense mechanism in plants, although in the last years there is increasing evidence on the role of SA in plant responses to abiotic stress. We recently reported that an increase in endogenous SA levels are able to counteract the inhibitory effects of several abiotic stress conditions during germination and seedling establishment of Arabidopsis thaliana and that this effect is modulated by gibberellins (GAs) probably through a member of the GASA (Giberellic Acid Stimulated in Arabidopsis) gene family, clearly showing the existence of a cross talk between these two plant hormones in Arabidopsis.Key words: abiotic stress responses, Arabidopsis thaliana, gibberellins, hormone cross-talk, salicylic acidGAs and SA play important roles in many processes of plant growth and development, and despite the recent papers reporting the existence of a complex network of hormone interactions, evidences of a cross talk between these two plant hormones have been very scarce.^1,2 These authors indicate that GAs are able to regulate SA biosynthesis during plant responses to pathogens. Interestingly, ABA has recently been proved to negative regulate SA-mediated defenses by downregulating SA biosynthesis.³ These data are consistent with the well known ABA/GAs antagonistic regulation of many aspects of plant development, such as seed dormancy or germination.^4,5 Thus, it seems clear that ABA and GAs are able to control plant immune responses by modulating the levels of salicylic acid and/or jasmonic acid.^1–3 In addition to the role of GAs in the regulation of plant responses to biotic stress, we have recently documented a role of GAs in early plant abiotic stress responses in Arabidopsis through modulation of SA levels,⁶ hormone that been involved in responses to abiotic stress conditions.⁷ For instance, it has been proved that SA has an important role in heat stress responses⁸ or in the improved germination of Arabidopsis thaliana seeds under salt stress conditions.⁹We showed that GAs and the overexpression of a GA-responsive gene were able to increase not only endogenous levels of SA, but also the expression of ics1 and npr1 genes, involved in SA biosynthesis and action, respectively.⁶ In addition, we have also analyzed expression levels of other genes that have been reported as SA-regulated. For instance, isocitrate lyase, a key enzyme involved in lipid metabolism during seed germination¹⁰ and a good marker of seed vigor under stress conditions,¹¹ was found to be induced by SA in germinated seeds of Arabidopsis thaliana.⁹ Thus, we proved that the expression of isocitrate lyase was upregulated in GASA4 overexpressing lines, and after exogenous application of GA₃ (Fig. 1), both situations increasing endogenous SA levels.⁶ We have documented that SA may have a role in some of the physiological processes associated with GAs, since exogenous application of SA was able to both revert the inhibitory effect of PCB on seed germination and improve germination of the GA-deficient mutant ga1–3.⁶ Thus, we can hypothesize that the GA-mediated induction of isocitrate lyase gene observed in Arabidopsis thaliana is the result of the increased levels of SA detected either after overexpression of the GA-induced GASA4 gene in Arabidopsis or after exogenous application of gibberellic acid. In other words, GAs are able to induce the expression of isocitrate lyase gene in a SA-dependent manner, producing the establishment of a vigorous seedling.⁹ These data support the idea that GAs may have an important role in SA biosynthesis and action, and that some of the physiological effects of this hormone may be mediate by SA. In summary, our results clearly show the existence of a cross talk between these two plant hormones during Arabidopsis thaliana seeds germination and early seedling growth under abiotic stress conditions, showing another junction in the complex mechanism of hormone interactions.Open in a separate windowFigure 1(A) Expression of the isocitrate lyase gene in FsGASA-overexpressing plants (G1 to G3) compared to Col-0. (B) Expression of the isocitrate lyase gene in Arabidopsis seedlings treated or not with 100 µM GA₃. mRNA levels were determined by northern blot analysis using total RNAs (10 µg/line) isolated from 7 d-old seedlings. Bottom, ethidium bromide stained gels showing rRNAs. Top: quantification of hybridization signals obtained by using a phosphoimage scanner. Data were normalized to the rRNA value. Blots were repeated twice and yielded similar results.     

Antioxidant responses of Propylaea japonica (Coleoptera: Coccinellidae) exposed to high temperature stress

Temperature is one of the most important environmental factors, and is responsible for a variety of physiological stress responses in organisms. Induced thermal stress is associated with elevated reactive oxygen species (ROS) generation leading to oxidative damage. The ladybeetle, Propylaea japonica (Thunberg) (Coleoptera: Coccinellidae), is considered a successful natural enemy because of its tolerance to high temperatures in arid and semi-arid areas in China. In this study, we investigated the effect of high temperatures (35, 37, 39, 41 and 43 °C) on the survival and activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidases (POD), glutathione-S-transferases (GST), and total antioxidant capacity (TAC) as well as malondialdehyde (MDA) concentrations in P. japonica adults. The results indicated that P. japonica adults could not survive at 43 °C. CAT, GST and TAC were significantly increased when compared to the control (25 °C), and this played an important role in the process of antioxidant response to thermal stress. SOD and POD activity, as well as MDA, did not differ significantly at 35 and 37 °C compared to the control; however, there were increased levels of SOD, POD and MDA when the temperature was above 37 °C. These results suggest that thermal stress leads to oxidative stress and antioxidant enzymes play important roles in reducing oxidative damage in P. japonica adults. This study represents the first comprehensive report on the antioxidant defense system in predaceous coccinellids (the third trophic level). The findings provide useful information for predicting population dynamics and understanding the potential for P. japonica as a natural enemy to control pest insects under varied environmental conditions.     

Growth of tomato (Lycopersicon esculentum) in response to salicylic acid under water stress

Abstract

Plants of Lycopersicon esculentum L. cv. K-25 were subjected to water stress by withholding water for 10 days at 20 (WS I) and 30 (WS II) days after sowing (DAS). Seedlings were sprayed with double distilled water (DDW) or 10^?5M salicylic acid (SA) at 45 DAS. The water stress at earlier stage of growth (20 day stage) was more inhibitory as compared to the later stage (30 day stage). The plants exposed to water stress exhibited a significant (p<0.05) decline in photosynthetic parameters, membrane stability index (MSI), leaf water potential, activity of nitrate reductase (NR), carbonic anhydrase (CA), chlorophyll and relative water content (RWC). A follow-up treatment with SA protected against the stress generated by water and significantly improved the above parameters. However, proline content and antioxidant enzymes increased under drought as well as under SA treatments.     

The physiological and biochemical responses to freezing stress of olive plants treated with salicylic acid

One-year-old olive (Olea europaea L. cv. Zard) plants were treated with 0.5, 1, and 2 mM salicylic acid (SA) and then exposed to nonfreezing and freezing temperatures (?5, ?10, and ?20°C) for 10 h. Untreated plants served as a control. Exposure to freezing temperatures caused a considerable increase in ion leakage and lipid peroxidation in olive leaves. Treatment with suitable exogenous SA (1.0 mM) prevented the increase in the ion leakage and lipid peroxidation caused by freezing temperatures, especially at ?5 and ?10°C. SA-induced freezing tolerance was accompanied by increased activities of antioxidant enzymes, such as guaiacol peroxidase, catalase, ascorbate peroxidase, and polyphenol oxidase, as compared to control plants. Proline, total phenolic content, and antioxidant capacity of olive leaves were declined significantly after exposure to freezing temperature, and their content decreased with lowering of freezing temperatures, while treatment with 1 mM SA induced a significant increase in their content. As a summary of these results, suitable concentration of SA (1 mM) could enhance freezing tolerance of olive plant by increasing antioxidant enzyme activities and decreasing MDA content through cell membrane integrity maintenance.