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1.  Effects of osmotic stress on antioxidant enzymes activities in leaf discs of PSAG12-IPT modified gerbera 
Leaf senescence is often caused by water deficit and the chimeric gene PSAG12-IPT is an auto-regulated gene delaying leaf senescence. Using in vitro leaf discs culture system, the changes of contents of chlorophylls, carotenoids, soluble protein and thiobarbituric acid reactive substance (TBARS) and antioxidant enzymes activities were investigated during leaf senescence of PSAGl2-IPT modified gerbera induced by osmotic stress compared with the control plant (wild type). Leaf discs were incubated in 20%, 40% (w/v) polyethylene glycol (PEG) 6 000 nutrient solution for 20 h under continuous light [130 µmol/(m2·s)]. The results showed that the contents of chlorophylls, carotenoids and soluble protein were decreased by osmotic stress with the decrease being more pronounced at 40% PEG, but that, at the same PEG concentration the decrease in the transgenic plants was significantly lower than that in the control plant. The activities of superoxide dismutase (SOD), catalases (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and dehydroascorbate reductase (DHAR) were stimulated by PEG treatment. However, the increases were higher in PSAG12-IPT transgenic plants than in the control plants, particularly at 40% PEG treatment. Lipid peroxidation (TBARS content) was increased by PEG treatment with the increase being much lower in transgenic plant than in the control plant. It could be concluded that the increases in the activities of antioxidant enzymes including SOD, CAT, APX, GPX and DHAR were responsible for the delay of leaf senescence induced by osmotic stress.
PMCID: PMC1906590  PMID: 17610324
Antioxidant enzymes; Gerbera; Leaf disc; Leaf senescence; Osmotic stress; PSAG12-IPT
2.  Creating Completely Both Male and Female Sterile Plants by Specifically Ablating Microspore and Megaspore Mother Cells 
Although genetically modified (GM) plants have improved commercially important traits, such as biomass and biofuel production, digestibility, bioremediation, ornamental value, and tolerance to biotic and abiotic stresses, there remain economic, political, or social concerns over potential ecological effects of transgene flow from GM plants. The current solution for preventing transgene flow from GM plants is genetically engineering sterility; however, approaches to generating both male and female sterility are limited. In addition, existing strategies for creating sterility lead to loss or modifications of entire flowers or floral organs. Here, we demonstrate that instead of the 1.5-kb promoter, the entire SOLO DANCERS (SDS) gene is required for its meiocyte-specific expression. We then developed an efficient method to specifically ablate microspore and megaspore mother cells using the SDS and BARNASE fusion gene, which resulted in complete sterility in both male and female reproductive organs in Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum), but did not affect plant growth or development, including the formation of all flower organs. Therefore, our research provides a general and effective tool to prevent transgene flow in GM plants.
PMCID: PMC4740954  PMID: 26870055
completely both male and female sterile plants; flower structure; genetic ablation; gene flow; microspore and megaspore mother cells; SOLO DANCERS
3.  Compound DNA vaccine encoding SAG1/ SAG3 with A2/B subunit of cholera toxin as a genetic adjuvant protects BALB/c mice against Toxoplasma gondii 
Parasites & Vectors  2013;6:63.
Intracellular parasites, such as T. gondii, present a plurality of antigens because of the complexity of its life cycle. Compound DNA vaccines bring a new approach and hope for the treatment of toxoplasmosis. In this study, a DNA vaccine encoding two major surface antigens SAG1, SAG3 from T. gondii, with A2/B subunit of cholera toxin as a genetic adjuvant was constructed.
BALB/c mice were immunized intramuscularly with PBS, pcDNA3.1, pSAG1, pSAG1/SAG3 and pSAG1/SAG3-CTXA2/B three times separately. Immunized mice were tested for IgG antibody and IFN-γ and IL-4 production by ELISA. The proliferation of T cells was measured by DNA synthesis assay and the lymphocyte subsets of spleen cells by flow cytometry. All the immunized mice were challenged with 103 highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally and the survival times were recorded.
An enhanced production of IgG antibodies, antigen-specific lymphocyte proliferation and IFN-γ production from splenic cells were induced in mice immunized with pSAG1/SAG3 compared to mice immunized with pSAG1 (P<0.05). Introduction of CTXA2/B further enhanced the Th1 cell-mediated immunity with higher levels of IFN-γ, lymphocyte proliferation activity and percentage of CD8+ T-cells. When challenged with lethal doses of T. gondii (1×103), all control mice (PBS and empty plasmid group) died within 6 days. Mice immunized with pSAG1 died within 8 days. While 20% and 40% survival rate were achieved from mice immunized with pSAG1/SAG3 and pSAG1/SAG3-CTXA2/B.
This study indicates the compound DNA vaccine encoding T. gondii antigens SAG1, SAG3 with CTXA2/B gene was a promising DNA vaccine candidate against toxoplasmosis, which could effectively enhance the humoral and cellular immune response and prolong survival time in vaccinated mice.
PMCID: PMC3622580  PMID: 23497561
Toxoplasma gondii; Surface antigen; SAG1; SAG3; CTXA2/B; DNA vaccination
4.  Elevated cytokinin content in ipt transgenic creeping bentgrass promotes drought tolerance through regulating metabolite accumulation 
Journal of Experimental Botany  2011;63(3):1315-1328.
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.
PMCID: PMC3276099  PMID: 22131157
Alanine; γ-aminobutyric acid (GABA); cytokinins; fructose; glycine; isopentyl transferase; maltose; metabolome; perennial grass; proline; ribose; senescence; senescence-activated promoter; sucrose; turfgrass; water stress
5.  Root-synthesized cytokinins improve shoot growth and fruit yield in salinized tomato (Solanum lycopersicum L.) plants 
Journal of Experimental Botany  2010;62(1):125-140.
Salinity limits crop productivity, in part by decreasing shoot concentrations of the growth-promoting and senescence-delaying hormones cytokinins. Since constitutive cytokinin overproduction may have pleiotropic effects on plant development, two approaches assessed whether specific root-localized transgenic IPT (a key enzyme for cytokinin biosynthesis) gene expression could substantially improve tomato plant growth and yield under salinity: transient root IPT induction (HSP70::IPT) and grafting wild-type (WT) shoots onto a constitutive IPT-expressing rootstock (WT/35S::IPT). Transient root IPT induction increased root, xylem sap, and leaf bioactive cytokinin concentrations 2- to 3-fold without shoot IPT gene expression. Although IPT induction reduced root biomass (by 15%) in control (non-salinized) plants, in salinized plants (100 mM NaCl for 22 d), increased cytokinin concentrations delayed stomatal closure and leaf senescence and almost doubled shoot growth (compared with WT plants), with concomitant increases in the essential nutrient K+ (20%) and decreases in the toxic ion Na+ (by 30%) and abscisic acid (by 20–40%) concentrations in transpiring mature leaves. Similarly, WT/35S::IPT plants (scion/rootstock) grown with 75 mM NaCl for 90 d had higher fruit trans-zeatin concentrations (1.5- to 2-fold) and yielded 30% more than WT/non-transformed plants. Enhancing root cytokinin synthesis modified both shoot hormonal and ionic status, thus ameliorating salinity-induced decreases in growth and yield.
PMCID: PMC2993914  PMID: 20959628
ABA; cytokinins; grafting; IPT; root zone temperature; root to shoot signalling; salinity; Solanum lycopersicum
6.  Photosynthesis, water use, and root viability under water stress as affected by expression of SAG12-ipt controlling cytokinin synthesis in Agrostis stolonifera 
Journal of Experimental Botany  2010;62(1):383-395.
Water stress reduces endogenous cytokinin (CK) content and may inhibit CK production. Maintenance of endogenous CK levels by genetic transformation with ipt in leaves and roots undergoing senescence may promote stress tolerance. This study was designed to determine the physiological effects of ipt expression on immature and mature leaves and in roots for plants exposed to different levels of water stress for creeping bentgrass (Agrostis stolonifera). Plants containing the ipt gene, encoding the enzyme adenine isopentenyl phosphotransferase for CK synthesis ligated to a senescence-activated promoter (SAG12), and wild-type ‘Penncross’ (WT) were grown hydroponically in a growth chamber and exposed to water stress by weekly additions of polyethylene glycol 8000 to reduce the growing solution osmotic potential from –0.05 to –0.3, –0.5, –0.7, –1.0, and –1.4 MPa. Immature and mature leaves and roots of SAG12-ipt creeping bentgrass were evaluated for ipt expression, CK content, leaf relative water content (RWC), chlorophyll content (Chl), photochemical efficiency (FvFm), osmotic adjustment (OA), photosynthesis rate (Pn), stomatal conductance (gs), transpiration (E), water use efficiency (WUE), carbon isotope discrimination (Δ), and root viability. Expression of ipt was detected in all plant parts and a higher CK content, primarily in the form of isopentyladenine (iPa), was found in SAG12-ipt plants but not in the WT plants under water stress. Immature leaves exhibited higher iPa and OA at all treatment levels. Mature leaves of SAG12-ipt plants maintained higher OA, Pn, Chl, WUE, and Δ, whereas gs and E were relatively unaffected compared to the WT. Roots of SAG12-ipt plants had higher levels of iPa and greater root viability than the WT. The results demonstrate that expression of ipt enhanced the tolerance of creeping bentgrass to water stress, which could be attributed to the positive effects on osmotic adjustment, efficient water use, and maintaining higher photosynthetic rate primarily for mature leaves, as well as increased root viability.
PMCID: PMC2993921  PMID: 20841349
Cytokinins; drought stress; osmotic stress; SAG12-ipt; senescence
7.  Anther-specific carbohydrate supply and restoration of metabolically engineered male sterility 
Journal of Experimental Botany  2010;61(10):2693-2706.
Male-sterile plants are used in hybrid breeding as well as for gene confinement for genetically modified plants in field trials and agricultural production. Apart from naturally occurring mutations leading to male sterility, biotechnology has added new possibilities for obtaining male-sterile plants, although so far only one system is used in practical breeding due to limitations in propagating male-sterile plants without segregations in the next generation or insufficient restoration of fertility when fruits or seeds are to be harvested from the hybrid varieties. Here a novel mechanism of restoration for male sterility is presented that has been achieved by interference with extracellular invertase activity, which is normally specifically expressed in the anthers to supply the developing microspores with carbohydrates. Microspores are symplastically isolated in the locular space of the anthers, and thus an unloading pathway of assimilates via the apoplasmic space is mandatory for proper development of pollen. Antisense repression of the anther-specific cell wall invertase or interference with invertase activity by expressing a proteinacious inhibitor under the control of the anther-specific invertase promoter results in a block during early stages of pollen development, thus causing male sterility without having any pleiotropic effects. Restoration of fertility was successfully achieved by substituting the down-regulated endogenous plant invertase activity by a yeast invertase fused to the N-terminal portion of potato-derived vacuolar protein proteinase II (PiII–ScSuc2), under control of the orthologous anther-specific invertase promoter Nin88 from tobacco. The chimeric fusion PiII–ScSuc2 is known to be N-glycosylated and efficiently secreted from plant cells, leading to its apoplastic location. Furthermore, the Nin88::PiII-ScSuc2 fusion does not show effects on pollen development in the wild-type background. Thus, such plants can be used as paternal parents of a hybrid variety, thereby the introgression of Nin88::PiII-ScSuc2 to the hybrid is obtained and fertility is restored. In order to broaden the applicability of this male sterility/restoration system to other plant species, a phylogenic analysis of plant invertases(β-fructofuranosidases) and related genes of different species was carried out. This reveals a specific clustering of the cell wall invertases with anther-specific expression for dicotyl species and another cluster for monocotyl plants. Thus, in both groups of plants, there seems to be a kind of co-evolution, but no recent common ancestor of these members of the gene family. These findings provide a helpful orientation to classify corresponding candidate genes in further plant species, in addition to the species analysed so far (Arabidopsis, tobacco, tomato, potato, carrots, rice, and wheat).
PMCID: PMC2882265  PMID: 20427415
Genetic engineering; hybrid breeding; invertase; male sterility; restoration
8.  Evaluation of protective immune responses induced by DNA vaccines encoding Toxoplasma gondii surface antigen 1 (SAG1) and 14-3-3 protein in BALB/c mice 
Parasites & Vectors  2012;5:273.
Toxoplasmosis, caused by an obligate intracellular protozoan parasite Toxoplasma gondii, has been a serious clinical and veterinary problem. Effective DNA vaccines against T. gondii can prevent and control the spread of toxoplasmosis, which is important for both human health and the farming industry. The T. gondii 14-3-3 protein has been proved to be antigenic and immunogenic and was a potential vaccine candidate against toxoplasmosis. In this study, we evaluated the immune responses induced by recombinant plasmids encoding T. gondii surface antigen 1 (SAG1) and 14-3-3 protein by immunizing BALB/c mice intramuscularly.
In the present study, BALB/c mice were randomly divided into five groups, including three experimental groups (pSAG1, p14-3-3 and pSAG1/14-3-3) and two control groups (PBS and pBudCE4.1), and were immunized intramuscularly three times. The levels of IgG antibodies and cytokine production in mouse sera were determined by enzyme-linked immunosorbent assays (ELISA). Two weeks after the last immunization, all mice were challenged intraperitoneally (i.p.) with 1×104 tachyzoites of T. gondii and the survival time of mice was observed and recorded every day.
Mice vaccinated with pSAG1, p14-3-3 or pSAG1/14-3-3 developed high levels of IgG2a and gamma interferon (IFN-γ) and low levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) compared to control groups (PBS or pBudCE4.1), which suggested a modulated Th1 type immune response (P<0.05). After intraperitoneal challenge with 1×104 tachyzoites of T. gondii (RH strain), the survival time of mice in experimental groups was longer than control groups (P<0.05). Mouse immunized with pSAG1/14-3-3 induced a higher level of IgG antibody response and significantly prolonged the survival time when compared with pSAG1 or p14-3-3 (P<0.05).
The study suggested that T. gondii 14-3-3 protein can induce effective immune responses in BALB/c mice and was a novel DNA vaccine candidate against toxoplasmosis, and the immune protective efficacy elicited by SAG1 gene was also demonstrated. Our results also showed multi-gene vaccine significantly enhanced immune responses and protective efficacy and was superior to the single-gene vaccine.
PMCID: PMC3547689  PMID: 23181694
Toxoplasma gondii; SAG1; 14-3-3; DNA vaccine; Immunity; BALB/c mice
9.  Protein accumulation in leaves and roots associated with improved drought tolerance in creeping bentgrass expressing an ipt gene for cytokinin synthesis 
Journal of Experimental Botany  2011;62(15):5311-5333.
Cytokinins (CKs) may be involved in the regulation of plant adaptation to drought stress. The objectives of the study were to identify proteomic changes in leaves and roots in relation to improved drought tolerance in transgenic creeping bentgrass (Agrostis stolonifera) containing a senescence-activated promoter (SAG12) and the isopentyl transferase (ipt) transgene that increases endogenous CK content. Leaves of SAG12-ipt bentgrass exhibited less severe senescence under water stress, as demonstrated by maintaining lower electrolyte leakage and lipid peroxidation, and higher photochemical efficiency (Fv/Fm), compared with the null transformant (NT) plants. SAG12-ipt plants had higher root/shoot ratios and lower lipid peroxidation in leaves under water stress than the NT plants. The suppression of drought-induced leaf senescence and root dieback in the transgenic plants was associated with the maintenance of greater antioxidant enzyme activities (superoxide dismutase, peroxidase, and catalase). The SAG12-ipt and NT plants exhibited differential protein expression patterns under well-watered and drought conditions in both leaves and roots. Under equivalent leaf water deficit (47% relative water content), SAG12-ipt plants maintained higher abundance of proteins involved in (i) energy production within both photosynthesis and respiration [ribulose 1,5-bisphosphate carboxylase (RuBisCO) and glyceraldehyde phosphate dehydrogenase (GAPDH)]; (ii) amino acid synthesis (methionine and glutamine); (iii) protein synthesis and destination [chloroplastic elongation factor (EF-Tu) and protein disulphide isomerases (PDIs)]; and (iv) antioxidant defence system (catalase and peroxidase) than the NT plants. These results suggest that increased endogenous CKs under drought stress may directly or indirectly regulate protein abundance and enzymatic activities involved in the above-mentioned metabolic processes, thereby enhancing plant drought tolerance.
PMCID: PMC3223035  PMID: 21831843
Cytokinins; drought stress; isopentyl transferase; perennial grass; proteome; senescence; senescence-activated promoter; turfgrass
10.  Regeneration of Transgenic Rice with Bacterial ipt Gene Driven by Senescence Specific (SAG12) Promoter by Particle Bombardment 
Transgenic rice plants were generated using particle bombardment to introduce the Agrobacterium cytokinin biosynthesis gene driven by Arabidopsis (Arabidopsis thaliana) senescence specific promoter (SAG12) for delaying leaf senescence. Using two plasmids we co-transformed one week old embryogenic calli derived from mature Japonica rice (Oryza sativa) variety Chin Guang. The selectable marker hygromycin phosphotransferase (hph) gene and the reporter gene B-ß-glucuronidase (uidA), both under the control of cauliflower mosaic virus (CaMV) 35S promoter were placed on the same co-integrate vector whereas the cytokinin biosynthesis gene, isopentenyl transferase (ipt) driven by the SAG12 promoter is supplied in another plasmid. A total of 32 transgenic rice plants were regenerated of which 27 plants were randomly selected for histochemical ß-glucuronidase (GUS) assay, PCR and Southern blot analysis. Co-integration frequencies of 88% and 69% were obtained for two linked genes (uidA and hph) and two unlinked genes (hph and ipt gene) respectively in R0 plants. Southern blot analysis confirmed the results of histochemical GUS assay and PCR amplifications. A complex integration pattern for all the transgenes including the multiple copies integration was predominantly observed.
PMCID: PMC3799406  PMID: 24575232
Co-transformation; Particle Bombardment; ipt Gene; SAG12 Promoter; Rice
11.  Restoring pollen fertility in transgenic male-sterile eggplant by Cre/loxp-mediated site-specific recombination system 
Genetics and Molecular Biology  2010;33(2):298-307.
This study was designed to control plant fertility by cell lethal gene Barnase expressing at specific developmental stage and in specific tissue of male organ under the control of Cre/loxP system, for heterosis breeding, producing hybrid seed of eggplant. The Barnase-coding region was flanked by loxP recognition sites for Cre-recombinase. The eggplant inbred/pure line (‘E-38') was transformed with Cre gene and the inbred/pure line (‘E-8') was transformed with the Barnase gene situated between loxp. The experiments were done separately, by means of Agrobacterium co-culture. Four T0 -plants with the Barnase gene were obtained, all proved to be male-sterile and incapable of producing viable pollen. Flowers stamens were shorter, but the vegetative phenotype was similar to wild-type. Five T 0 -plants with the Cre gene developed well, blossomed out and set fruit normally. The crossing of male-sterile Barnase-plants with Cre expression transgenic eggplants resulted in site-specific excision with the male-sterile plants producing normal fruits. With the Barnase was excised, pollen fertility was fully restored in the hybrids. The phenotype of these restored plants was the same as that of the wild-type. Thus, the Barnase and Cre genes were capable of stable inheritance and expression in progenies of transgenic plants.
PMCID: PMC3036876  PMID: 21637486
eggplant; male sterility; Barnase gene; Cre gene; Cre/loxP system
12.  Delayed expression of SAGs correlates with longevity in CMS wheat plants compared to its fertile plants 
Reproductive sinks regulate monocarpic senescence in crop plants. Monocarpic senescence was studied in wheat fertile (cv. HW 2041) and its isonuclear cytoplasmic male sterile (CMS) line. CMS plants exhibited slower rate of senescence accompanied by longer green leaf area duration and slower deceleration in chlorophyll, protein content, PN and rubisco content coupled with lower protease activities than fertile (F) plants. CMS plants also exhibited lower ROS levels and less membrane damage than F plants. CMS plants maintained better antioxidant defense, less oxidative damage in chloroplast and higher transcript levels of both rbcL and rbcS genes during senescence than F plants. F plants exhibited early induction and higher expression of SAGs like serine and cysteine proteases, glutamine synthetases GS1 and GS2, WRKY53 transcription factor and decline in transcript levels of CAT1 and CAT2 genes than CMS plants. Hence, using genetically fertile and its CMS line of wheat it is confirmed that delayed senescence in the absence of reproductive sinks is linked with slower protein oxidation, rubisco degradation and delayed activation of SAGs. Better antioxidant defense in chloroplasts at later stages of senescence was able to mitigate the deleterious effects of ROS in CMS plants. We propose that delayed increase in ROS in cytoplasmic male sterile wheat plants resulted in delayed activation of WRKY53, SAGs and the associated biochemical changes than fertile plants.
PMCID: PMC3988327  PMID: 24757323
Senescence; Oxidative stress; Protein carbonylation; CMS wheat
13.  Analysis of the Maize dicer-like1 Mutant, fuzzy tassel, Implicates MicroRNAs in Anther Maturation and Dehiscence 
PLoS ONE  2016;11(1):e0146534.
Sexual reproduction in plants requires development of haploid gametophytes from somatic tissues. Pollen is the male gametophyte and develops within the stamen; defects in the somatic tissues of the stamen and in the male gametophyte itself can result in male sterility. The maize fuzzy tassel (fzt) mutant has a mutation in dicer-like1 (dcl1), which encodes a key enzyme required for microRNA (miRNA) biogenesis. Many miRNAs are reduced in fzt, and fzt mutants exhibit a broad range of developmental defects, including male sterility. To gain further insight into the roles of miRNAs in maize stamen development, we conducted a detailed analysis of the male sterility defects in fzt mutants. Early development was normal in fzt mutant anthers, however fzt anthers arrested in late stages of anther maturation and did not dehisce. A minority of locules in fzt anthers also exhibited anther wall defects. At maturity, very little pollen in fzt anthers was viable or able to germinate. Normal pollen is tricellular at maturity; pollen from fzt anthers included a mixture of unicellular, bicellular, and tricellular pollen. Pollen from normal anthers is loaded with starch before dehiscence, however pollen from fzt anthers failed to accumulate starch. Our results indicate an absolute requirement for miRNAs in the final stages of anther and pollen maturation in maize. Anther wall defects also suggest that miRNAs have key roles earlier in anther development. We discuss candidate miRNAs and pathways that might underlie fzt anther defects, and also note that male sterility in fzt resembles water deficit-induced male sterility, highlighting a possible link between development and stress responses in plants.
PMCID: PMC4706427  PMID: 26745722
14.  Proteomic changes associated with expression of a gene (ipt) controlling cytokinin synthesis for improving heat tolerance in a perennial grass species 
Journal of Experimental Botany  2010;61(12):3273-3289.
Cytokinins (CKs) are known to regulate leaf senescence and affect heat tolerance, but mechanisms underlying CK regulation of heat tolerance are not well understood. A comprehensive proteomic study was conducted to identify proteins altered by the expression of the adenine isopentenyl transferase (ipt) gene controlling CK synthesis and associated with heat tolerance in transgenic plants for a C3 perennial grass species, Agrostis stolonifera. Transgenic plants with two different inducible promoters (SAG12 and HSP18) and a null transformant (NT) containing the vector without ipt were exposed to 20 °C (control) or 35 °C (heat stress) in growth chambers. Two-dimensional electrophoresis and mass spectrometry analysis were performed to identify protein changes in leaves and roots in response to ipt expression under heat stress. Transformation with ipt resulted in protein changes in leaves and roots involved in multiple functions, particularly in energy metabolism, protein destination and storage, and stress defence. The abundance levels of six leaf proteins (enolase, oxygen-evolving enhancer protein 2, putative oxygen-evolving complex, Rubisco small subunit, Hsp90, and glycolate oxidase) and nine root proteins (Fd-GOGAT, nucleotide-sugar dehydratase, NAD-dependent isocitrate dehydrogenase, ferredoxin-NADP reductase precursor, putative heterogeneous nuclear ribonucleoprotein A2, ascorbate peroxidase, dDTP-glucose 4–6-dehydratases-like protein, and two unknown proteins) were maintained or increased in at least one ipt transgenic line under heat stress. The diversity of proteins altered in transgenic plants in response to heat stress suggests a regulatory role for CKs in various metabolic pathways associated with heat tolerance in C3 perennial grass species.
PMCID: PMC2905195  PMID: 20547565
Cytokinins; high temperature; HSP18-ipt; SAG12-ipt; senescence
15.  Water-Deficit Inducible Expression of a Cytokinin Biosynthetic Gene IPT Improves Drought Tolerance in Cotton 
PLoS ONE  2013;8(5):e64190.
Water-deficit stress is a major environmental factor that limits agricultural productivity worldwide. Recent episodes of extreme drought have severely affected cotton production in the Southwestern USA. There is a pressing need to develop cotton varieties with improved tolerance to water-deficit stress for sustainable production in water-limited regions. One approach to engineer drought tolerance is by delaying drought-induced senescence via up-regulation of cytokinin biosynthesis. The isopentenyltransferase gene (IPT) that encodes a rate limiting enzyme in cytokinin biosynthesis, under the control of a water-deficit responsive and maturation specific promoter PSARK was introduced into cotton and the performance of the PSARK::IPT transgenic cotton plants was analyzed in the greenhouse and growth chamber conditions. The data indicate that PSARK::IPT-transgenic cotton plants displayed delayed senescence under water deficit conditions in the greenhouse. These plants produced more root and shoot biomass, dropped fewer flowers, maintained higher chlorophyll content, and higher photosynthetic rates under reduced irrigation conditions in comparison to wild-type and segregated non-transgenic lines. Furthermore, PSARK::IPT-transgenic cotton plants grown in growth chamber condition also displayed greater drought tolerance. These results indicate that water-deficit induced expression of an isopentenyltransferase gene in cotton could significantly improve drought tolerance.
PMCID: PMC3651191  PMID: 23675526
16.  Tapetum and middle layer control male fertility in Actinidia deliciosa 
Annals of Botany  2013;112(6):1045-1055.
Background and Aims
Dioecism characterizes many crop species of economic value, including kiwifruit (Actinidia deliciosa). Kiwifruit male sterility occurs at the microspore stage. The cell walls of the microspores and the pollen of the male-sterile and male-fertile flowers, respectively, differ in glucose and galactose levels. In numerous plants, pollen formation involves normal functioning and degeneration timing of the tapetum, with calcium and carbohydrates provided by the tapetum essential for male fertility. The aim of this study was to determine whether the anther wall controls male fertility in kiwifruit, providing calcium and carbohydrates to the microspores.
The events occurring in the anther wall and microspores of male-fertile and male-sterile anthers were investigated by analyses of light microscopy, epifluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL assay) and transmission electron microscopy coupled with electron spectroscopy. The possibility that male sterility was related to anther tissue malfunctioning with regard to calcium/glucose/galactose provision to the microspores was also investigated by in vitro anther culture.
Key Results
Both tapetum and the middle layer showed secretory activity and both degenerated by programmed cell death (PCD), but PCD was later in male-sterile than in male-fertile anthers. Calcium accumulated in cell walls of the middle layer and tapetum and in the exine of microspores and pollen, reaching higher levels in anther wall tissues and dead microspores of male-sterile anthers. A specific supply of glucose and calcium induced normal pollen formation in in vitro-cultured anthers of the male-sterile genotype.
The results show that male sterility in kiwifruit is induced by anther wall tissues through prolonged secretory activity caused by a delay in PCD, in the middle layer in particular. In vitro culture results support the sporophytic control of male fertility in kiwifruit and open the way to applications to overcome dioecism and optimize kiwifruit production.
PMCID: PMC3783237  PMID: 23965617
Actinidia deliciosa; anther; anther culture; calcium; dioecism; flower male fertility flower male sterility; glucose; middle layer; pollen; programmed cell death; tapetum
17.  The NAC-like gene ANTHER INDEHISCENCE FACTOR acts as a repressor that controls anther dehiscence by regulating genes in the jasmonate biosynthesis pathway in Arabidopsis  
Journal of Experimental Botany  2013;65(2):621-639.
ANTHER INDEHISCENCE FACTOR (AIF), a NAC-like gene, was identified in Arabidopsis. In AIF:GUS flowers, β-glucuronidase (GUS) activity was detected in the anther, the upper parts of the filaments, and in the pollen of stage 7–9 young flower buds; GUS activity was reduced in mature flowers. Yellow fluorescent protein (YFP)+AIF-C fusion proteins, which lacked a transmembrane domain, accumulated in the nuclei of the Arabidopsis cells, whereas the YFP+AIF fusion proteins accumulated in the membrane and were absent in the nuclei. Further detection of a cleaved AIF protein in flowers revealed that AIF needs to be processed and released from the endoplasmic reticulum in order to function. The ectopic expression of AIF-C caused a male-sterile phenotype with indehiscent anthers throughout flower development in Arabidopsis. The presence of a repressor domain in AIF and the similar phenotype of indehiscent anthers in AIF-C+SRDX plants suggest that AIF acts as a repressor. The defect in anther dehiscence was due to the down-regulation of genes that participate in jasmonic acid (JA) biosynthesis, such as DAD1/AOS/AOC3/OPR3/OPCL1. The external application of JA rescued the anther indehiscence in AIF-C and AIF-C+SRDX flowers. In AIF-C+VP16 plants, which are transgenic dominant-negative mutants in which AIF is converted to a potent activator via fusion to a VP16-AD motif, the anther dehiscence was promoted, and the expression of DAD1/AOS/AOC3/OPR3/OPCL1 was up-regulated. Furthermore, the suppression of AIF through an antisense strategy resulted in a mutant phenotype similar to that observed in the AIF-C+VP16 flowers. The present data suggest a role for AIF in controlling anther dehiscence by suppressing the expression of JA biosynthesis genes in Arabidopsis.
PMCID: PMC3904717  PMID: 24323506
Anther dehiscence; ANTHER INDEHISCENCE FACTOR; jasmonate signalling; NAC-like gene; repressor.
18.  Transformation and analysis of tobacco plant var Petit havana with T-urf13 gene under anther-specific TA29 promoter 
3 Biotech  2011;1(2):73-82.
T-urf13, a well-documented cms-associated gene from maize, has been shown to render methomyl sensitivity to heterologous systems like rice, yeast and bacteria when expressed constitutively. Since these transgenic plants were fertile, it was hypothesized that T-urf13 gene if expressed in anthers may result in male sterility that could be used for hybrid seed production. Hence, this work was aimed at analysing whether T-urf13 gene when expressed in anthers can result in male sterile plants or requires methomyl treatment to cause male sterility (controllable). This is the first report of transformation of tobacco with T-urf13 gene under anther-specific promoter (TA29) with or without mitochondrial targeting sequence. Most of the transgenic plants obtained were fertile; this was surprising as many male sterile plants were expected as T-urf13 gene is a cms associated gene. Our results suggest that it may not be possible to obtain male sterility by expressing URF13 in the anther by itself or by methomyl application.
PMCID: PMC3339608  PMID: 22582148
cms-T maize; Hybrid seeds; Male sterility; Tobacco transformation; T-urf13; URF13; Chemistry; Biomaterials; Bioinformatics; Cancer Research; Stem Cells; Agriculture; Biotechnology
19.  Transformation and analysis of tobacco plant var Petit havana with T-urf13 gene under anther-specific TA29 promoter 
3 Biotech  2011;1(2):73-82.
T-urf13, a well-documented cms-associated gene from maize, has been shown to render methomyl sensitivity to heterologous systems like rice, yeast and bacteria when expressed constitutively. Since these transgenic plants were fertile, it was hypothesized that T-urf13 gene if expressed in anthers may result in male sterility that could be used for hybrid seed production. Hence, this work was aimed at analysing whether T-urf13 gene when expressed in anthers can result in male sterile plants or requires methomyl treatment to cause male sterility (controllable). This is the first report of transformation of tobacco with T-urf13 gene under anther-specific promoter (TA29) with or without mitochondrial targeting sequence. Most of the transgenic plants obtained were fertile; this was surprising as many male sterile plants were expected as T-urf13 gene is a cms associated gene. Our results suggest that it may not be possible to obtain male sterility by expressing URF13 in the anther by itself or by methomyl application.
PMCID: PMC3339608  PMID: 22582148
cms-T maize; Hybrid seeds; Male sterility; Tobacco transformation; T-urf13; URF13
20.  Biparental inheritance of plastidial and mitochondrial DNA and hybrid variegation in Pelargonium 
Molecular Genetics and Genomics   2009;282(6):587-593.
Plastidial (pt) and mitochondrial (mt) genes usually show maternal inheritance. Non-Mendelian, biparental inheritance of plastids was first described by Baur (Z Indukt Abstamm Vererbungslehre 1:330–351, 1909) for crosses between Pelargonium cultivars. We have analyzed the inheritance of pt and mtDNA by examining the progeny from reciprocal crosses of Pelargoniumzonale and P. inquinans using nucleotide sequence polymorphisms of selected pt and mt genes. Sequence analysis of the progeny revealed biparental inheritance of both pt and mtDNA. Hybrid plants exhibited variegation: our data demonstrate that the inquinans chloroplasts, but not the zonale chloroplasts bleach out, presumably due to incompatibility of the former with the hybrid nuclear genome. Different distribution of maternal and paternal sequences could be observed in different sectors of the same leaf, in different leaves of the same plant, and in different plants indicating random segregation and sorting-out of maternal and paternal plastids and mitochondria in the hybrids. The substantial transmission of both maternal and paternal mitochondria to the progeny turns Pelargonium into a particular interesting subject for studies on the inheritance, segregation and recombination of mt genes.
PMCID: PMC2777209  PMID: 19787375
Mitochondria; Chloroplasts; Biparental inheritance; Heteroplasmy; Pelargonium
21.  Regulated Expression of a Cytokinin Biosynthesis Gene IPT Delays Leaf Senescence and Improves Yield under Rainfed and Irrigated Conditions in Canola (Brassica napus L.) 
PLoS ONE  2015;10(1):e0116349.
Delay of leaf senescence through genetic modification can potentially improve crop yield, through maintenance of photosynthetically active leaves for a longer period. Plant growth hormones such as cytokinin regulate and delay leaf senescence. Here, the structural gene (IPT) encoding the cytokinin biosynthetic enzyme isopentenyltransferase was fused to a functionally active fragment of the AtMYB32 promoter and was transformed into canola plants. Expression of the AtMYB32xs::IPT gene cassette delayed the leaf senescence in transgenic plants grown under controlled environment conditions and field experiments conducted for a single season at two geographic locations. The transgenic canola plants retained higher chlorophyll levels for an extended period and produced significantly higher seed yield with similar growth and phenology compared to wild type and null control plants under rainfed and irrigated treatments. The yield increase in transgenic plants was in the range of 16% to 23% and 7% to 16% under rainfed and irrigated conditions, respectively, compared to control plants. Most of the seed quality parameters in transgenic plants were similar, and with elevated oleic acid content in all transgenic lines and higher oil content and lower glucosinolate content in one specific transgenic line as compared to control plants. The results suggest that by delaying leaf senescence using the AtMYB32xs::IPT technology, productivity in crop plants can be improved under water stress and well-watered conditions.
PMCID: PMC4300212  PMID: 25602960
22.  Transcriptomes of the Anther Sporophyte: Availability and Uses 
Plant and Cell Physiology  2011;52(9):1459-1466.
An anther includes sporophytic tissues of three outer cell layers and an innermost layer, the tapetum, which encloses a locule where the gametophytic microspores mature to become pollen. The sporophytic tissues also comprise some vascular cells and specialized cells of the stomium aligning the long anther axis for anther dehiscence. Studies of the anther sporophytic cells, especially the tapetum, have recently expanded from the use of microscopy to molecular biology and transcriptomes. The available sequencing technologies, plus the use of laser microdissection and in silico subtraction, have produced high-quality anther sporophyte transcriptomes of rice, Arabidopsis and maize. These transcriptomes have been used for research discoveries and have potential for future discoveries in diverse areas, including developmental gene activity networking and changes in enzyme and metabolic domains, prediction of protein functions by quantity, secretion, antisense transcript regulation, small RNAs and promoters for generating male sterility. We anticipate that these studies with rice and other transcriptomes will expand to encompass other plants, whose genomes will be sequenced soon, with ever-advancing sequencing technologies. In comprehensive gene activity profiling of the anther sporophyte, studies involving transcriptomes will spearhead investigation of the downstream gene activity with proteomics and metabolomics.
PMCID: PMC3172567  PMID: 21743085
Anther; Anther development; Anther transcripts; Sporophyte transcripts; Tapetum; Transcriptomes
23.  Male reproductive development: gene expression profiling of maize anther and pollen ontogeny 
Genome Biology  2008;9(12):R181.
During flowering, central anther cells switch from mitosis to meiosis, ultimately forming pollen containing haploid sperm. Four rings of surrounding somatic cells differentiate to support first meiosis and later pollen dispersal. Synchronous development of many anthers per tassel and within each anther facilitates dissection of carefully staged maize anthers for transcriptome profiling.
Global gene expression profiles of 7 stages representing 29 days of anther development are analyzed using a 44 K oligonucleotide array querying approximately 80% of maize protein-coding genes. Mature haploid pollen containing just two cell types expresses 10,000 transcripts. Anthers contain 5 major cell types and express >24,000 transcript types: each anther stage expresses approximately 10,000 constitutive and approximately 10,000 or more transcripts restricted to one or a few stages. The lowest complexity is present during meiosis. Large suites of stage-specific and co-expressed genes are identified through Gene Ontology and clustering analyses as functional classes for pre-meiotic, meiotic, and post-meiotic anther development. MADS box and zinc finger transcription factors with constitutive and stage-limited expression are identified.
We propose that the extensive gene expression of anther cells and pollen represents the key test of maize genome fitness, permitting strong selection against deleterious alleles in diploid anthers and haploid pollen. Because flowering plants show a substantial bias for male-sterile compared to female-sterile mutations, we propose that this fitness test is general. Because both somatic and germinal cells are transcriptionally quiescent during meiosis, we hypothesize that successful completion of meiosis is required to trigger maturation of anther somatic cells.
PMCID: PMC2646285  PMID: 19099579
24.  A transfer RNAArg gene of Pelargonium chloroplasts, but not a 5S RNA gene, is efficiently transcribed after injection into Xenopus oocyte nuclei. 
Nucleic Acids Research  1984;12(21):8253-8268.
We present the primary structure of a chloroplast tRNAArgACG gene of the plant, Pelargonium zonale, and its faithful expression in Xenopus oocyte nuclei. This tRNAArg gene is located 250 bp downstream of a 5S RNA gene within a cloned 5kb long ribosomal DNA segment (Fig. 1). The Pelargonium tRNAArg gene shares 97% and 86% sequence homology with tRNAArgACG genes of Spirodela oligorhiza and Euglena gracilis chloroplasts, respectively, and also extensive homology (70%) with the corresponding gene of E. coli. It lacks an intervening sequence and, like eukaryotic tRNA genes, does not code for the 3' terminal CCA nucleotides. Moreover, the chloroplast tRNAArg gene carries all the sequence elements essential for transcription by vertebrate RNA polymerase III since it is efficiently expressed in Xenopus oocyte nuclei, even in the presence of 1 microgram/ml alpha-amanitin. In Xenopus oocyte nuclei, no transcripts of the chloroplast 5S RNA gene were detected.
PMCID: PMC320309  PMID: 6209611
25.  AMS-dependent and independent regulation of anther transcriptome and comparison with those affected by other Arabidopsis anther genes 
BMC Plant Biology  2012;12:23.
In flowering plants, the development of male reproductive organs is controlled precisely to achieve successful fertilization and reproduction. Despite the increasing knowledge of genes that contribute to anther development, the regulatory mechanisms controlling this process are still unclear.
In this study, we analyzed the transcriptome profiles of early anthers of sterile mutants aborted microspores (ams) and found that 1,368 genes were differentially expressed in ams compared to wild type anthers, affecting metabolism, transportation, ubiquitination and stress response. Moreover, the lack of significant enrichment of potential AMS binding sites (E-box) in the promoters of differentially expressed genes suggests both direct and indirect regulation for AMS-dependent regulation of anther transcriptome involving other transcription factors. Combining ams transcriptome profiles with those of two other sterile mutants, spl/nzz and ems1/exs, expression of 3,058 genes were altered in at least one mutant. Our investigation of expression patterns of major transcription factor families, such as bHLH, MYB and MADS, suggested that some closely related homologs of known anther developmental genes might also have similar functions. Additionally, comparison of expression levels of genes in different organs suggested that anther-preferential genes could play important roles in anther development.
Analysis of ams anther transcriptome and its comparison with those of spl/nzz and ems1/exs anthers uncovered overlapping and distinct sets of regulated genes, including those encoding transcription factors and other proteins. These results support an expanded regulatory network for early anther development, providing a series of hypotheses for future experimentation.
PMCID: PMC3305669  PMID: 22336428

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