Elevated cytokinin content in ipt transgenic creeping bentgrass promotes drought tolerance through regulating metabolite accumulation

Increased endogenous plant cytokinin (CK) content through transformation with an adenine isopentyl transferase (ipt) gene has been associated with improved plant drought tolerance. The objective of this study is to determine metabolic changes associated with elevated CK production in ipt transgenic creeping bentgrass (Agrostis stolonifera L.) with improved drought tolerance. Null transformants (NTs) and plants transformed with ipt controlled by a stress- or senescence-activated promoter (SAG12-ipt) were exposed to well-watered conditions or drought stress by withholding irrigation in an environmental growth chamber. Physiological analysis confirmed that the SAG12-ipt line (S41) had improved drought tolerance compared with the NT plants. Specific metabolite changes over the course of drought stress and differential accumulation of metabolites in SAG12-ipt plants compared with NT plants at the same level of leaf relative water content (47% RWC) were identified using gas chromatography-mass spectroscopy. The metabolite profiling analysis detected 45 metabolites differentially accumulated in response to ipt expression or drought stress, which included amino acids, carbohydrates, organic acids, and organic alcohols. The enhanced drought tolerance of SAG12-ipt plants was associated with the maintenance of accumulation of several metabolites, particularly amino acids (proline, γ-aminobutyric acid, alanine, and glycine) carbohydrates (sucrose, fructose, maltose, and ribose), and organic acids that are mainly involved in the citric acid cycle. The accumulation of these metabolites could contribute to improved drought tolerance due to their roles in the stress response pathways such as stress signalling, osmotic adjustment, and respiration for energy production.     

Protein profile analysis of salt-responsive proteins in leaves and roots in two cultivars of creeping bentgrass differing in salinity tolerance

Knowledge of stress-responsive proteins is critical for further understanding the molecular mechanisms of stress tolerance. The objectives of this study were to establish a proteomic map for a perennial grass species, creeping bentgrass (A. stolonifera L.), and to identify differentially expressed, salt-responsive proteins in two cultivars differing in salinity tolerance. Plants of two cultivars (‘Penncross’ and ‘Penn-A4’) were irrigated daily with water (control) or NaCl solution to induce salinity stress in a growth chamber. Salinity stress was obtained by adding NaCl solution of 2, 4, 6, and 8 dS m⁻¹ in the soil daily for 2-day intervals at each concentration, and then by watering soil with 10 dS m⁻¹ solution daily for 28 days. For proteomic map, using two-dimensional electrophoresis (2-DE), approximately 420 and 300 protein spots were detected in leaves and roots, respectively. A total of 148 leaf protein spots and 40 root protein spots were excised from the 2-DE gels and subjected to mass spectrometry analysis. In total, 106 leaf protein spots and 24 root protein spots were successfully identified. Leaves had more salt-responsive proteins than roots in both cultivars. The superior salt tolerance in ‘Penn-A4’, indicated by shoot extension rate, relative water content, and cell membrane stability during the 28-day salinity stress could be mainly associated with its higher level of vacuolar H⁺-ATPase in roots and UDP-sulfoquinovose synthase, methionine synthase, and glucan exohydrolase in leaves, as well as increased accumulation of catalase and glutathione S-transferase in leaves. Our results suggest that salinity tolerance in creeping bentgrass could be in part controlled by an alteration of ion transport through vacuolar H⁺-ATPase in roots, maintenance of the functionality and integrity of thylakoid membranes, sustained polyamine biosynthesis, and by the activation of cell wall loosening proteins and antioxidant defense mechanisms.     

Overexpression of Arabidopsis ABF3 gene confers enhanced tolerance to drought and heat stress in creeping bentgrass

The Arabidopsis, abscisic acid responsive element-binding factor 3, ABF3 is known to play an important role in stress responses via regulating the expression of stress-responsive genes. In this study, we introduced pCAMBIA3301 vector harboring the ABF3 gene into creeping bentgrass (Agrostis stolonifera) through Agrobacterium-mediated transformation in order to develop a stress-tolerant variety of turfgrass. After transformation, putative transgenic plants were selected using the herbicide resistance assay. Genomic integration of the transgene was confirmed by genomic PCR and Southern blot analysis, and gene expression was validated by northern blot analysis. Under drought-stressed condition, the transgenic plants overexpressing ABF3 displayed significantly enhanced drought tolerance with higher water content and slower water loss rate than the control plants. Furthermore, the stomata of the ABF3 transgenic plants closed more than those of wild-type creeping bentgrass plants, under both non-stressed and ABA treatment conditions. In addition, the transgenic plants showed enhanced tolerance to heat stress. These results suggest that the overexpression of the ABF3 gene in creeping bentgrass might enhance survival in water-limiting and high temperature environments through increased stomatal closure and reduced water losses.     

Photosynthetic acclimation to high temperatures associated with heat tolerance in creeping bentgrass

Photosynthetic responses to increasing temperatures play important roles in regulating heat tolerance. The objectives of this study were to determine photosynthetic acclimation to increasing temperatures for creeping bentgrass (Agrostis stolonifera L.) and to examine changes in major photosynthetic components (photosynthetic pigments, photochemical efficiency, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activity, and activation state of rubisco) involved in heat responses of photosynthesis. 'Penncross' was exposed to 20, 25, 30, and 35 degrees C for 7d at each temperature (acclimated) before being exposed to 40 degrees C for 28d or directly exposed to 40 degrees C for 28d from 20 degrees C (non-acclimated) in growth chambers. Leaf net photosynthetic rate (Pn), photochemical efficiency, rubisco activity, rubisco activation state, chlorophyll content, and carotenoid content decreased when grasses were subjected to severe heat stress at 40 degrees C for 28d. The declines in rubisco activity and activation state were most dramatic among different photosynthetic components examined in this study. Heat-acclimated plants were able to maintain significantly higher Pn, the content of chlorophyll and carotenoid, and the level of rubisco activity and activation state during subsequent exposure to severe heat stress, compared to non-acclimated plants. These results suggested that photosynthetic acclimation to increasing temperatures contributed to creeping bentgrass tolerance to severe heat stress, which was associated with the maintenance of both higher light-harvesting capacity and carbon fixation activity during heat stress.     

Antioxidative responses in roots and shoots of creeping bentgrass under high temperature: effects of nitrogen and cytokinin

It has been previously reported that either nitrogen (N) or cytokinin (CK) applications can alleviate heat stress injury on creeping bentgrass, with some studies reporting enhanced antioxidant metabolism being related to stress protection. The objective of this research was to investigate the simultaneous effects of CK and N on the antioxidant enzyme activity and isoforms of heat stressed creeping bentgrass. 'L-93' creeping bentgrass treated with three rates of CK (trans-zeatin riboside, tZR, 0, 10 and 100μM, designated by CK0, 10, and 100) and two nitrogen rates (2.5 and 7.5kgNha(-1) biweekly, low and high N) in a complete factorial arrangement was maintained in a 38/28°C (day/night) growth chamber for 28d and then harvested. Grass grown at high N (averaged across CK rates) had higher O(2)(-) production, H(2)O(2) concentration, and malondialdehyde content in roots. The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) in roots were enhanced 19%, 22%, and 24%, respectively, by high N relative to low N. Twenty-eight days of heat stress resulted in either the development of new isoforms or enhanced isoform intensities of SOD, APX, and POD in roots compared to plant responses prior to heat stress. However, no apparent differences were observed across treatments. Both SOD and POD showed different isoform patterns between roots and shoots, suggesting the function of these isoforms could be tissue specific. Interestingly, no CK effects on these antioxidant parameters were found in this experiment. These results demonstrate the impacts of N on antioxidant metabolism of creeping bentgrass under heat stress with some differences between roots and shoots, but no simultaneous impacts of CK and N.     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Differential accumulation of proteins in leaves and roots associated with heat tolerance in two Kentucky bluegrass genotypes differing in heat tolerance

To understand the mechanisms of heat stress responses in perennial grasses, differential proteins in leaves and roots of two genotypes of Kentucky bluegrass (Poa pratensis), including heat-tolerant ‘Midnight’ and heat-sensitive ‘Brilliant’, were analyzed with two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). Plants were exposed to heat stress for 28 days in growth chambers. Under 7–28 days of heat stress, leaf photochemical efficiency declined significantly while electrolyte leakage increased in leaves and roots, and to a lesser extent for heat-tolerant ‘Midnight’ than for heat-sensitive ‘Brilliant’. Compared with leaves, cell membrane damage due to heat stress was more severe in roots. The 2-DE and MS analysis identified 37 heat-responsive proteins in leaves, 28 heat-responsive proteins in roots; 14 proteins in leaves and 9 proteins in roots exhibited differential expression between the two genotypes. The results indicate that proteins involved in metabolism and energy in leaves and those in antioxidant defense in roots are associated with heat tolerance in Kentucky bluegrass. The differential accumulation of these proteins might be the reason for different heat tolerance in two Kentucky bluegrass genotypes in aerial and underground parts.     

Overexpression of barley <Emphasis Type="Italic">hva1</Emphasis> gene in creeping bentgrass for improving drought tolerance

The objectives of this study were to test the feasibility of introducing barley hva1 gene, a LEA3 member, into perennial grass species using the Agrobacterium-mediated transformation technique and to determine whether heterologous expression of hva1 would alleviate water-deficit injury in grass species. Creeping bentgrass (Agrostis stolonifera var. palustris), a drought-intolerant grass species, was transformed transiently or stably using three different promoters in conjunction with the downstream report/target genes. Two abscisic acid (ABA)-inducible promoters, ABA1 and ABA2 derived from ABA-response complex (ABRC3) were used to examine stress-responsive expression of the green fluorescent protein (GFP). Transient expression of GFP demonstrated the inducibility of ABA1 and ABA2 promoters in response to exogenous ABA application. The ABA2 promoter was further studied for stress-responsive expression of hva1 and a maize Ubi-1 promoter was tested for constitutive expression of the gene. In the T₀ generation, the Ubi-1::hva1 transformants displayed variable expression levels of HVA1 protein under normal growth conditions. The hva1 gene in the ABA2::hva1 transformants maintained low expression under well-watered conditions, but was upregulated under water-deficit conditions. The tolerance to water deficit of T₀ transgenic lines was assessed by measuring leaf relative water content and visually rating the severity of leaf wilting during to water stress. Under water-stressed conditions, some transgenic lines maintained high water content in leaves and showed significantly less extent of leaf wilting compared with non-transgenic control plants. These results indicated that the introduction of barley hva1 gene using constitutive or stress-inducible promoters lessened water-deficit injury in creeping bentgrass, suggesting that heterologous expression of LEA3 protein genes may enhance the survival ability of creeping bentgrass in water limiting environments.     

Accumulation of terpenoid phytoalexins in maize roots is associated with drought tolerance

Maize (Zea mays) production, which is of global agro‐economic importance, is largely limited by herbivore pests, pathogens and environmental conditions, such as drought. Zealexins and kauralexins belong to two recently identified families of acidic terpenoid phytoalexins in maize that mediate defence against both pathogen and insect attacks in aboveground tissues. However, little is known about their function in belowground organs and their potential to counter abiotic stress. In this study, we show that zealexins and kauralexins accumulate in roots in response to both biotic and abiotic stress including, Diabrotica balteata herbivory, Fusarium verticillioides infection, drought and high salinity. We find that the quantity of drought‐induced phytoalexins is positively correlated with the root‐to‐shoot ratio of different maize varieties, and further demonstrate that mutant an2 plants deficient in kauralexin production are more sensitive to drought. The induction of phytoalexins in response to drought is root specific and does not influence phytoalexin levels aboveground; however, the accumulation of phytoalexins in one tissue may influence the induction capacity of other tissues.     

Metabolic pathways regulated by abscisic acid,salicylic acid and γ‐aminobutyric acid in association with improved drought tolerance in creeping bentgrass (Agrostis stolonifera)

Abscisic acid (ABA), salicylic acid (SA) and γ‐aminobutyric acid (GABA) are known to play roles in regulating plant stress responses. This study was conducted to determine metabolites and associated pathways regulated by ABA, SA and GABA that could contribute to drought tolerance in creeping bentgrass (Agrostis stolonifera). Plants were foliar sprayed with ABA (5 μM), GABA (0.5 mM) and SA (10 μM) or water (untreated control) prior to 25 days drought stress in controlled growth chambers. Application of ABA, GABA or SA had similar positive effects on alleviating drought damages, as manifested by the maintenance of lower electrolyte leakage and greater relative water content in leaves of treated plants relative to the untreated control. Metabolic profiling showed that ABA, GABA and SA induced differential metabolic changes under drought stress. ABA mainly promoted the accumulation of organic acids associated with tricarboxylic acid cycle (aconitic acid, succinic acid, lactic acid and malic acid). SA strongly stimulated the accumulation of amino acids (proline, serine, threonine and alanine) and carbohydrates (glucose, mannose, fructose and cellobiose). GABA enhanced the accumulation of amino acids (GABA, glycine, valine, proline, 5‐oxoproline, serine, threonine, aspartic acid and glutamic acid) and organic acids (malic acid, lactic acid, gluconic acid, malonic acid and ribonic acid). The enhanced drought tolerance could be mainly due to the enhanced respiration metabolism by ABA, amino acids and carbohydrates involved in osmotic adjustment (OA) and energy metabolism by SA, and amino acid metabolism related to OA and stress‐defense secondary metabolism by GABA.     

Lipoxygenase activity and proline accumulation in leaves and roots of olive trees in response to drought stress

The olive tree ( Olea europaea L.) is commonly grown in the Mediterranean basin and is able to resist severe and prolonged drought. Levels of proline (PRO) and malondialdehyde (MDA), and the lipoxygenase (LOX) activity were determined in 2-year-old olive plants (cv. 'Coratina') grown in environmental conditions characterized by high temperatures and high photosynthetic photon flux density levels and gradually subjected to a controlled water deficit for 20 days. Before and during the experimental period, leaf and root samples were collected and analysed for PRO and MDA. The levels of PRO increased in parallel with the severity of drought stress in both leaves and roots. Significant increases of LOX activity and MDA content were also observed during the progressive increment of drought stress in both leaf and root tissues. Measurements of transpiration and photosynthetic rate, stomatal conductance and substomatal CO₂ concentration were carried out during the experiment. The accumulation of PRO indicates a possible role of PRO in drought tolerance. The increases of MDA content and LOX activity show that the water deficit is associated with lipid peroxidation mechanisms.     

Protein synthesis in mitochondria purified from roots, leaves and flowers of sugar beet

Highly purified, intact and functional mitochondria were isolated from roots and leaves of a number of fertile and male-sterile lines of sugar beet ( Beta vulgaris L.). Intact and functional mitochondria were successfully isolated from the flowers of fertile plants, but not from the flowers of male-sterile plants. Several alternative methods for the homogenization of male-sterile flowers were tried. Their failure suggests that the mitochondria from male-sterile flowers are more sensitive to mechanical damage than mitochondria from fertile, or other organs of male-sterile, plants.
In organello protein synthesis was optimized with respect to the total concentration of amino acids, the concentration of [³⁵S]-methionine, pH and respiratory substrate. Inhibitor experiments showed that the mitochondrial preparations contained mitochondrial translational activity only. With the exception of one band, no processing or proteolytic breakdown in either root or leaf mitochondrial protein synthesis products could be detected in pulse-chase experiments. Submitochondrial fractionation experiments showed the presence of two soluble polypeptides, whereas all other polypeptides were membrane bound.
The polypeptide patterns of root, leaf and flower mitochondria were very similar with the exception of 4 polypeptides involved in glycine oxidation. These 4 polypeptides were present in large amounts in leaf mitochondria and just detectable in flower mitochondria. The patterns of polypeptides syntesized in mitochondria isolated from roots, leaves and flowers also showed a number of organ-specific differences. Six qualitative and 6 quantitative differences were found between mitochondria isolated from these three organs. No unique polypeptides were found to be synthesized either by flower mitochondria or by mitochondria from roots and leaves of male-sterile plants compared to their male-fertile counterparts.     

Transgenic creeping bentgrass plants expressing a <Emphasis Type="Italic">Picea wilsonii</Emphasis> dehydrin gene (<Emphasis Type="Italic">PicW</Emphasis>) demonstrate improved freezing tolerance

Agrostis stolonifera L. ‘Penn A-4’ is a common creeping bentgrass species that is widely used in urban landscaping and golf courses. To prolong the green stage of this grass, a dehydrin gene PicW isolated from Wilson’s spruce (Picea wilsonii) was transformed into plants of ‘Penn A-4’ cultivar via a straightforward stolon node infection system. A putative transgenic plant was obtained and its tolerance to low-temperature stress was evaluated. When the transgenic line was subjected to a freezing (??5 °C) treatment, it showed better viability and more robust physiology than wild type, as evidenced by higher soluble sugar and proline contents, and lower relative electrical conductivity and malondialdehyde content. The transgenic line also showed tolerance to a chilling treatment (5 °C), although its performance was not significantly different from that of wild-type plants. Overall, the research here clearly revealed the explicit role of PicW in increasing freezing tolerance of grass at the whole-plant level, and demonstrated that the straightforward stolon node transformation method could be well used to genetically modify turfgrass. The obtained transgenic line might be as genetic resource for breeding program and practiced to grow in cold temperate zones.     

Enhancement of heavy metal tolerance and accumulation efficiency by expressing Arabidopsis ATP sulfurylase gene in alfalfa

Abstract

Transgenic alfalfa (Medicago sativa L.) plants overexpressing the Arabidopsis ATP sulfurylase gene were generated using Agrobacterium-mediated genetic transformation to enhance their heavy metal accumulation efficiency. The ATP sulfurylase gene was cloned from Arabidopsis, following exposure to vanadium (V) and lead (Pb), and transferred into an Agrobacterium tumefaciens binary vector. This was co-cultivated with leaf explants of the alfalfa genotype Regen SY. Co-cultivated leaf explants were cultured on callus and somatic embryo induction medium, followed by regeneration medium for regenerating complete transgenic plants. The transgenic nature of the plants was confirmed using PCR and southern hybridization. The expression of Arabidopsis ATP sulfurylase gene in the transgenic plants was evaluated through RT-PCR. The selected transgenic lines showed increased tolerance to a mixture of five heavy metals and also demonstrated enhanced metal uptake ability under controlled conditions. The transgenic lines were fertile and did not exhibit any apparent morphological abnormality. The results of this study indicated an effective approach to improve the heavy metal accumulation ability of alfalfa plants which can then be used for the remediation of contaminated soil in arid regions.