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??-Glucuronidase activity during development of male gametophyte from transgenic and nontransgenic plants
Abstract
Beta-glucuronidase (GUS) was histochemically analyzed in anthers and pollen of potato, tobacco and tomato. GUS activity was determined in transgenic plants containing a chimaeric GUS gene and in untransformed plants. In anthers of transgenic plants at premeiotic and meiotic stages of sporogenous development, indigogenic precipitation indicative of GUS activity was consistently manifest in cells of the vascular cylinder, the connectivum and the stomium while no activity was found in the tapetal and sporogenous tissues. At similar stages, anther sections of untransformed plants did not show any indigo blue staining. At later stages of microspore and pollen development, anthers of both transgenic and untransformed plants demonstrated consistently high levels of GUS activity in tapetal and sporogenic cells. In anthers of transgenic plants, GUS was also present in the vascular cylinder, the connectivum and the stomium. These results indicate that in anthers of transgenic potato, tobacco and tomato the chimaeric GUS gene product was localized tissue specifically. They also show that an endogenous GUS gene was expressed in a temporal- and spatial-specific manner in the tapetum and pollen of both transformed and untransformed plants.
... Analysis of the expression of promoter-gus fusions showed that the chs promoter drives expression only in the tapetum cell layer and in the outer parenchymatic cells of the connectivum . The CaMV 35s promoter on the other hand shows expression in almost every cell type of the anther except for the tapetum and sporogenous tissues (Plegt and Bino, 1989). The principal site of flavonoid synthesis in the anther is the tapetum (Herdt et al., 1978;Kehrel and Wiermann, 1985;Beerhueset al., 1989), from which the pigments are transported into the locule and incorporated in the outer surface of the pollen grain (Wiermann and Vieth, 1983). ...
... These results might be explained by assuming that the expression driven by the CaMV 35s promoter is either relatively low or exhibits a different temporal and/or spatial regulation compared with the endogenous chs gene expression in anthers. Plegt and Bino (1989) showed that the CaMV 35s-driven expression pattern of the gus gene in anthers is diffuse and that the tapetum cells exhibit no activity. Because the tapetum cells are the principal site of flavonoid biosynthesis in anthers (Kehrel and Wiermann, 1985;Beerhues et al., 1989), we aimed at modification of the cell-type specific expression of the strong CaMV 35s promoter to achieve antisense inhibition of anther pigmentation. ...
... Promoter deletion analysis revealed, however, that the presence of this anther box within the chs promoter is not a prerequisite for anther-specific gene expression, suggesting that additional sequences within the minimal promoter may direct the organ-specific expression (van der Meer et al., 1990). Plegt and Bino (1989) previously showed that the CaMV 35S promoter drives expression in almost all cell types of the anther except for the tapetal and sporogenous tissues. We confirmed this result ( Figure 4) and showed that introduction of the chsA anther box sequence in the CaMV 35S promoter altered this expression pattern: the tapetal cell layer showed GUS activity superimposed on the basic CaMV-gus staining pattern. ...
Inhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers, presumably because the viral CaMV 35S promoter was insufficiently expressed in cell types of this organ in which the pigments are produced. Insertion of the anther box, a homologous sequence found in other genes expressed in anthers, resulted in a modified expression pattern driven by this promoter, as monitored by the beta-glucuronidase (gus) gene. In addition to the basic CaMV 35S expression pattern in anthers, GUS activity was observed in tapetum cells when the modified promoter was fused to the gus gene. This promoter construct was subsequently used to drive an antisense chs gene in transgenic petunia, which led to the inhibition of pigment synthesis in anthers of five of 35 transformants. Transgenic plants with white anthers were male sterile due to an arrest in male gametophyte development. This finding indicated that flavonoids play an essential role in male gametophyte development.
... three different GUS genes have been identified in Arabidopsis thaliana: AtGUS1, AtGUS2 and AtGUS3 [42]. Further studies have confirmed the wide diffusion of GUSs in plants [35,[43][44][45][46][47] and the presence of enzymatic activity in all the different organs [40,[48][49][50][51][52][53][54][55][56][57][58][59]. ...
... Concerning the GUS role in plants, their involvement in the changes of polysaccharide moieties [40,60] or in the release of signal molecules [50,57,[61][62][63] has been proposed. In particular, it has been demonstrated that GUSs are involved in regulating the glycosylation degree of arabinogalactan proteins [60] and in the changes in cell wall composition associated with cell elongation [40,60]. ...
Gene duplication played a fundamental role in eukaryote evolution and different copies of a given gene can be present in extant species, often with expressions and functions differentiated during evolution. We assume that, when such differentiation occurs in a gene copy, this may be indicated by its maintenance in all the derived species. To verify this hypothesis, we compared the histological expression domains of the three β-glucuronidase genes (AtGUS) present in Arabidopsis thaliana with the GUS evolutionary tree in angiosperms. We found that AtGUS gene expression overlaps in the shoot apex, the floral bud and the root hairs. In the root apex, AtGUS3 expression differs completely from AtGUS1 and AtGUS2, whose transcripts are present in the root cap meristem and columella, in the staminal cell niche, in the epidermis and in the proximal cortex. Conversely, AtGUS3 transcripts are limited to the old border-like cells of calyptra and those found along the protodermal cell line. The GUS evolutionary tree reveals that the two main clusters (named GUS1 and GUS3) originate from a duplication event predating angiosperm radiation. AtGUS3 belongs to the GUS3 cluster, while AtGUS1 and AtGUS2, which originate from a duplication event that occurred in an ancestor of the Brassicaceae family, are found together in the GUS1 cluster. There is another, previously undescribed cluster, called GUS4, originating from a very ancient duplication event. While the copy of GUS4 has been lost in many species, copies of GUS3 and GUS1 have been conserved in all species examined.
... This enzymatic activity is also reported in maize developing kernels (Muhitch, 1998). Moreover, endogenous GUSB activity is present in many other plants such as fruit wall, seed coat, endosperm, embryos (Hu et al., 1990) and during male gametophyte development of potato, tobacco, and tomato (Plegt and Bino, 1989). ...
... Following research on plant GUSB or "endogenous" GUSB that plays key roles in development of the primary leaves of rye plant (Secale cereale), the same is found to be in the pedicel of maize kernels during its developing and lignin biosynthesis (Schulz and Weissenböck, 1987;Anhalt and Weissenböck, 1992;Muhitch, 1998). Endogenous GUSB activity in plant is reported in fruit wall, seed coat, endosperm, embryos, and during male gametophyte development of many plants (Plegt and Bino, 1989;Hu et al., 1990). Inhibition of endogenous GUSB activity leads to seedling developmental problems such as arresting the growth and inhibiting root-hair development in tobacco (Nicotiana tabacum) and parasitic angiosperm, Cuscuta pentagona (Sudan et al., 2006;Schoenbeck et al., 2007). ...
... Despite the extensive efforts that have been devoted to the study of plant promoters, we found no promoter that was characterized as being expressed highly in all tissues except those associated with male reproduction. In the absence of such information, we proposed the best candidate to be an enhanced 35S Cauliflower Mosaic Virus (CaMV) promoter, given that expression of this promoter was shown to be lacking in the tapetum and sporogenous cells of developing anthers in dicots (Plegt and Bino, 1989). Furthermore, in maize transformed with a glyphosate tolerance gene driven by an enhanced 35S promoter, male reproductive tissues were the only tissue type reported to be unprotected from the herbicide in mature plants sprayed with glyphosate (Feng et al., 2014). ...
Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δatp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δatp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δatp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δatp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.
... Despite the extensive efforts that have been devoted to the study of plant promoters, we found no promoter that was characterized as being expressed highly in all tissues except those associated with male reproduction. In the absence of such information, we proposed the best candidate to be an enhanced CaMV 35S promoter, given that expression of this promoter was shown to be lacking in the tapetum and sporogenous cells of developing anthers in dicots (Plegt and Bino, 1989). Furthermore, in maize transformed with a glyphosate tolerance gene driven by an enhanced 35S promoter, male reproductive tissues were the only tissue type reported to be unprotected from the herbicide in mature plants sprayed with glyphosate (Feng, et al., 2014). ...
Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Datp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Datp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Datp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Datp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.
... Heparanase, a heparan sulfate degrading endoglycosidase, is overexpressed in almost all cancer types where it is closely associated with tumorigenesis and metastasis [19]. Furthermore, glycosidases have been reported to function as important gene markers, for example, the gene encoding β-galactosidase, lacZ, is extensively used as a reporter gene in animals and yeast [20]. Glycosidases also play a critical role in industry with xylanases and cellulases of particular interest for the bulk production of bioethanol [21]. ...
Carbohydrates are an essential class of biomolecule and carbohydrate active enzymes (CAZys) catalyze their synthesis, refinement, and degradation, hence contributing an overall regulatory capacity to their underpinning physiological roles. Here we survey recent accomplishments for accessing defined carbohydrate structures, suitably equipped with FRET probe capability, followed by their utilization in studying particular classes of CAZy.
... In most studies, the sGUS activities in plants have been reported under acidic conditions (pH 4.0−6.0; Sood 1980; Plegt and Bino 1989;Hu et al. 1990;Alwen et al. 1992;Hodal et al. 1992;Wozniak and Owens 1994;Sudan et al. 2006;Schoenbeck et al. 2007). However, the sGUS from S. baicalensis is different, because its pH optimum is between 6.0 and 7.0, and the activity completely disappeared at pH values lower than 4.5 (Marimoto et al. 1995). ...
The study examined the relationship between the contents of principal flavones of Scutellaria baicalensis Georgi and the activity of endogenous b-glucuronidase (sGUS) in differentiated and undifferentiated in vitro cultures grown on solid and liquid medium. Using RP-HPLC, it was shown that sGUS activity did not correlate with the increase in biomass of cultures, but depended on tissue differentiation. In hairy roots cultured both on liquid and solid medium, it was ten times higher than in undifferentiated cultures. At the same time, the share of aglycone forms in the total content of flavones during the culture cycle depended on the growing conditions. It was significantly higher on agar medium than on liquid medium. A correlation between sGUS activity and total aglycone content, as well as wogonin content, was observed only in hairy roots grown in the liquid medium.
... However, our initial experiments with sorghum indicated that GUS reporters would not be ideal, due to positive GUS staining in control, non-transformed pollen grains ( Supplementary Fig. 1). Other published reports also indicate that GUS activity is detectable in the male gametophyte of a number of plant species 3,4 , spurring our labs to use green fluorescent protein (GFP)-based reporter plasmids as an alternative. For these experiments, we chose plasmids with promoters known to be highly expressed in grass pollen (maize Zm13, rice Actin1 or maize ZmUbiquitin1; refs. ...
... 19 Furthermore, glycosidases have been reported to function as important gene markers, for example, the gene encoding b-galactosidase, lacZ, is extensively used as a reporter gene in animals and yeast. 20 Glycosidases also play a critical role in industry with xylanases and cellulases of particular interest for the bulk production of bio-ethanol. 21 This review aims to survey the development and application of fluorescent probes to detect this important class of enzyme using appropriately labelled carbohydrate substrates (from 2010 onwards). ...
Carbohydrates are an essential class of biomolecule and carbohydrate active enzymes (CAZys) catalyse their synthesis, refinement, and degradation, hence contributing an overall regulatory capacity to their underpinning physiological roles. As such, there is a considerable current requirement to be able to monitor, quantify and inhibit CAZy activity. It is here that Förster resonance energy transfer (FRET) is emerging as a powerful tool in enabling this, through synthetic conjugation of appropriate fluorogenic partners into a particular CAZy substrate. In this review we survey recent accomplishments in synthetic methodology for accessing defined carbohydrate structures, suitably equipped with FRET probe capability, followed by their utilisation in studying particular classes of CAZy. The majority of examples concern endo-acting glycosidases, but emergent probes for exo-glycosidases, glycosyl transferases and catalytic antibodies are also examined.
... However, initial experiments with sorghum indicated that GUS reporters would not be ideal, due to positive GUS staining in control, non-transformed pollen grains (Supplemental Figure 1). Other published reports also indicate that GUS activity is detectable in the male gametophyte of a number of plant species 3,4 , spurring our labs to use green fluorescent protein (GFP)-based reporter plasmids as an alternative. For these experiments, we chose plasmids with promoters known to be highly expressed in grass pollen (maize Zm13, rice Actin1, or maize ZmUbiquitin1). ...
The development of rapid and efficient transformation methods for many plant species remains an obstacle in both the basic and applied plant sciences. A novel method described by Zhao et al. (2017) used magnetic nanoparticles to deliver DNA into pollen grains of several dicot species, and one monocot (lily), to achieve transformation (“pollen magnetofection”). Using the published protocol, extensive trials by two independent research groups showed no indication of transient transformation success with pollen from two monocots, maize and sorghum. To further address the feasibility of magnetofection, lily pollen was used for side-by-side trials of magnetofection with a proven methodology for transient transformation, biolistics. Using a Green Fluorescent Protein reporter plasmid, transformation efficiency with the biolistic approach averaged 0.7% over three trials. However, the same plasmid produced no recognizable transformants via magnetofection, despite screening >3500 individual pollen grains. We conclude that pollen magnetofection is not effective for transient transformation of pollen for at least three species of monocots, and suggest that efforts to replicate the magnetofection protocol in dicot species would be useful to fully assess its potential.
ARISING FROM Zhao et al. Nature Plants https://doi.org/10.1038/s41477-017-0063-z (2017)
... As expected the uidA-gene was not detected in any of them (Table 3). Hence this is assumed to be an endogenous activity in Petunia seeds, which − to our knowledge − has not yet been reported for Petunia, but for various other seedlings [39][40][41][42][43]. Thus, GUS-staining could not be used as reliable marker for plastid transmission but only for preselection of events. ...
As already demonstrated in greenhouse trials, outcrossing of transgenic plants can be drastically reduced via transgene integration into the plastid. We verified this result in the field with Petunia, for which the highest paternal leakage has been observed. The variety white 115 (W115) served as recipient and Pink Wave (PW) and the transplastomic variant PW T16, encoding the uidA reporter gene, as pollen donor. While manual pollination in the greenhouse led to over 90% hybrids for both crossings, the transgenic donor resulted only in 2% hybrids in the field. Nevertheless paternal leakage was detected in one case which proves that paternal inheritance of plastid-located transgenes is possible under artificial conditions. In the greenhouse, paternal leakage occurred in a frequency comparable to published results. As expected natural pollination reduced the hybrid formation in the field from 90 to 7.6% and the transgenic donor did not result in any hybrid.
... In order to further evidence the toxicity of MazF in plant cells, a double transformation strategy was used. As reported in the literature, the CaMV 35S promoter drives expression in almost all plant cell types except in the tapetal and sporogenous tissues of anthers [Plegt and Bino, 1989]. Therefore, to combine a protective expression of mazE in all plant tissues except tapetum with a toxic expression of mazF in the tapetum, transformation of the TA29-mazF construct was performed in a 35S-mazE background. ...
Background/aims:
The Escherichia coli MazF is an endoribonuclease that cleaves mRNA at ACA sequences, thereby triggering inhibition of protein synthesis. The aim of this study is to evaluate the efficiency of the mazEF toxin-antitoxin system in plants to develop biotechnological tools for targeted cell ablation.
Methods:
A double transformation strategy, combining expression of the mazE antitoxin gene under the control of the CaMV 35S promoter, reported to drive expression in all plant cells except within the tapetum, together with the expression of the mazF gene under the control of the TA29 tapetum-specific promoter in transgenic tobacco, was applied.
Results:
No transgenic TA29-mazF line could be regenerated, suggesting that the TA29 promoter is not strictly tapetum specific and that MazF is toxic for plant cells. The regenerated 35S-mazE/TA29-mazF double-transformed lines gave a unique phenotype where the tapetal cell layer was necrosed resulting in the absence of pollen.
Conclusion:
These results show that the E. colimazEF system can be used to induce death of specific plant cell types and can provide a new tool to plant cell ablation.
... In contrast, the expression of CP4-EPSPS protein from a construct with the rice actin 1 gene promoter was relatively low in the leaf but high in whole anther and pollen (Heck et al. 2005;Feng et al. 2010a; cassette 1, Fig. 6.1a). These findings are consistent with previous studies showing lack of CaMV 35S promoter activity in the tapetum cell layer and microspore mother cells, which are critical for male reproductive functions (Plegt and Bino 1989). These two promoters, therefore, have complementary activities which together generate the desired expression profile for whole-plant tolerance over a wide developmental window. ...
Weeds are one of the major causes of yield loss and, depending on the weed pressure and methods of weed control, their yields can be reduced up to 50 %. Agricultural biotechnology has enabled engineering of crops to make them tolerant to one or more broad-spectrum herbicides, allowing for the in-crop use of those herbicides for simplified but effective weed control. The incorporation of herbicide-tolerant crops into farming systems further facilitates no-tillage or low-tillage practices by controlling emerging weeds through post-planting herbicide application, leading to increased water retention in the soil, less soil disruption, less soil erosion, and increased organic matter in the soil. This review discusses the development of glyphosate- and glufosinate-tolerant biotech traits, and the use of trait stacking to fill the immediate needs of farmers facing the challenges of hard-to-control and resistant weeds in their fields. This discussion also considers the development of an additional herbicide-trait system for dicamba tolerance.
... . PCR and endogenous GUS activity analysis demonstrated their expression in all the different organs (Sood, 1980;Schulz and Weissenböck, 1987;Plegt and Bino, 1989;Hu et al., 1990;Alwen et al., 1992;Anhalt and Weissenböck, 1992;Wozniak and Owens, 1994;Morimoto et al., 1995;Morimoto et al., 1998;Muhitch, 1998;Sudan et al., 2006;Schoenbeck et al., 2007;Woo et al., 2007). ...
A new highly sensitive whole-mount in situ hybridization method, based on tyramide signal amplification (TSA-MISH) was developed and a combined GFP detection and TSA-MISH procedure was applied for the first time in plants, to precisely define the spatial pattern of AtGUS1 and AtGUS2 expression in the root apex. β-glucuronidases (GUSs) belonging to the glycosyl hydrolases (GHs) 79 family, are widely distributed in plants, but their functional role has not yet been fully investigated. In the model system Arabidopsis Thaliana, three different AtGUS genes have been identified which encode proteins with putative different fates. Endogenous GUS expression has been detected in different organs and tissues, but the cyto-histological domains of gene expression remain unclear. The results here reported show co-expression of AtGUS1 and AtGUS2 in different functional zones of the root apex (the cap central zone, the root cap meristem, the staminal cell niche and the cortical cell layers of the proximal meristem), while AtGUS2 is exclusively expressed in the cap peripheral layer and in the epidermis in the elongation zone. Interestingly, both genes are not expressed in the stelar portion of the proximal meristem. A spatial (cortex vs. stele) and temporal (proximal meristem vs. transition zone) regulation of AtGUS1 and AtGUS2 expression is therefore active in the root apex. This expression pattern, although globally consistent with the involvement of GUS activity in both cell proliferation and elongation, clearly indicates that AtGUS1 and AtGUS2 could control distinct downstream process depending on the developmental context and the interaction with other players of root growth control. In the future, the newly developed approaches may well be very useful to dissect such interactions.
... Whether untransformed potato plants exhibit endogenous GUS or GUS-like activities is still unclear. Plegt & Bino (1989) and Wenzler et al. (1989) reported a GUS activity in various potato tissues, while our previous work (DeTurck et al., 2002) in agreement with Jefferson et al. (1987) did not. ...
Transgenesis developed in the last 20 years offers new possibilities for crop protection. The transgenic process, however, requires the use of marker fusion genes to select and visualize the transformed tissues. Although the expression products of these marker genes are stably expressed in crops, little attention has been given to assess the eventual risks of these recombinant proteins on phytophage populations. Three independent transgenic potato (Solanum tuberosum) clones from the cultivar Désirée (DG5, DG18, and DG20) carrying the commonly used nptII-gus gene construct and exhibiting different β-glucuronidase activity (0.843 ± 0.011, 0.576 ± 0.096, and 0.002 ± 0.000 pmol min-1.mg-1, respectively) were evaluated to determine the impact of the encoded proteins on the behaviour, development, reproduction, and demography of the peach-potato aphid, Myzus persicae, under laboratory-controlled light and temperature. Our results revealed that the transgenic event can alter aphid physiology or behaviour. Experiments showed a probiotic effect of one transgenic line, the DG5, resulting in reduced prereproductive period and mortality, and enhanced daily fecundity, which was expressed in a greater population growth potential (rm = 0.205 vs. rm = 0.174 of the control). In contrast, aphids fed with the DG18 line exhibited reduced adult survival and reproductive period but no alteration of their demographic parameters (rm = 0.176). Finally, no physiological alteration was induced in aphids fed on a DG20 diet (rm = 0.170). Behavioural experiments conducted in a 4-choice olfactometer demonstrated that insects were significantly more attracted by the odour of transgenic DG18 potato plant than that of Désirée non-transformed plant, spending twice as much time in the DG18 plant odour. The two other transformed clones (DG5 and DG20) were as attractive as the non-transformed cultivar. It is concluded that the β-glucuronidase expression in potato plants might be responsible for the probiotic effect measured on the feeding aphids, whereas alteration of the foliage odour would result from a pleiotropic effect.
... quantified, or histochemically determined (Jefferson, 1987; Jefferson et al., 1987). Since plant metabolic and structural activities can affect foreign gene expression (Plegt and Bino, 1989), or compounds inhibiting foreign enzyme activity can be present (Fromm et al., 1987), we analyzed the applicability of GUS and CAT reporter genes to carnation transformation . A study of the factors in carnation leaf extract responsible for the inhibition of GUS but not CAT activity is presented. ...
The applicability of β- glucuronidase and chloramphenicol acetyltransferase reporter genes to a carnation (Dianthus caryophyllus L.) transformation procedure, was analyzed. Transgenic tobacco (Nicotiana tabacum L.) plants expressing the respective reporter genes were prepared and used as the enzyme source. Carnation leaf extract strongly inhibited enzymatic activity of β- glucuronidase, but not that of chloramphenicol acetyltransferase. One or more carnation phenolic compounds, acting in a noncompetitive manner, is suggested as the cause of the observed inhibition of fluorometrically assayed β- glucuronidase activity. This inhibition was eliminated by treating the carnation leaf extract with polyvinylpolypyrrolidone.
... The CaMV 35S promoter has been shown to be expressed in the vascular tissue, connective, and wall layers but not in the tapetum (Plegt and Bino, 1989;van der Meer et al., 1992). Therefore, it is unlikely that TAZ1 expression under the control of the CaMV 35S promoter could have triggered cosuppression directly in the tapetal cells. ...
TAZ1 (TAPETUM DEVELOPMENT ZINC FINGER PROTEIN1 ; renamed from PEThy; ZPT3-2) cDNA was first isolated as an anther-specific cDNA from petunia. Here, we report a functional characterization that includes analysis of spatial and temporal expression profiles and examination of anther phenotypes in TAZ1-silenced plants. TAZ1 showed a bi-phasic expression pattern. In the premeiotic phase, TAZ1 transcripts were found to accumulate in all cell types of the anther except the tapetum and gametophytic tissues, whereas the postmeiotic phase of anther development was characterized by expression exclusively in the tapetum. Silencing of TAZ1 by cosuppression resulted in aberrant development and precocious degeneration of the tapetum, followed by extensive microspore abortion that started soon after their release from pollen tetrads. A few pollen grains that survived showed reduced flavonol accumulation, defects in pollen wall formation, and poor germination rates. This study demonstrates an essential role for TAZ1 in the postmei-otic phase of tapetum development.
... Intrinsic GUS activity is absent from most plant tissues, although some low-level glucuronidase activity has been described for several plant species or may be present in bacteria contaminating soil-grown plants. GUS activity from some bacterial contaminations can be specifically inhibited when the appropriate assay conditions are used (Plegt and Bino, 1989;Kosugi et al., 1990;Alwen et al., 1992). When GUS substrates are used that produce fluorescent products, there are actually two background problems: (1) non-specific cleavage of the probe resulting in non-specific fluorescent staining of the tissue and (2) fluorescence of common plant cell components (Figs. 3 B1 and 3 B2). ...
: The detailed analysis of the expression pattern of a plant gene can give important clues about its function in plant development, cell differentiation and defence reactions. Gene expression studies have been greatly facilitated by the employment of proteins like β-glucuronidase (GUS), green fluorescent protein (GFP), and firefly luciferase (LUC) as reporters of gene activity. The application of reporter genes in plants, specifically in the field of gene expression studies, has expanded over the years from a mere tool to quantify (trans) gene expression in tissue samples, to real-time imaging of in planta promoter dynamics. To correctly interpret the activity that is given by each reporter, it is important to have a good understanding of the intrinsic properties of the different reporter proteins. Here we discuss those properties of GUS, LUC and GFP that are of interest in gene expression studies.
... The expression of PR-la by vira1 infection and SA treatment has been studied for more than 10 years, yet an understanding Severa1 studies have documented GUS artifacts associated with the histochemical GUS activity assay (Plegt and Bino, 1989;Mascarenhas and Hamilton, 1992;Tor et al., 1992). In RNA was isolated from the -903 T 3 lines indicated. ...
Pathogenesis-related protein-la (PR-la) is a protein of unknown function that is strongly induced during the onset of systemic acquired resistance (SAR) in tobacco. The expression of PR-la is under complex regulation that is controlled at least partially by the rate of transcription. In this study, we demonstrated that 661 bp of 5'flanking DNA was sufficient to impart tobacco mosaic virus and salicylic acid inducibility to a reporter gene. The PR-la promoter did not respond significantly to treatments with either auxin or cytokinin. Experiments with the protein synthesis inhibitor cycloheximide indicated that protein synthesis is required for salicylate-dependent mRNA accumulation. At flowering, the PR-la gene was expressed primarily in the mesophyll and epidermal tissues of the leaf blade and the sepals of the flower. Several artifacts, most importantly ectopic expression in pollen, were associated with the use of the P-glucuronidase reporter gene.
... However, in anthers from stage seven [25], both enhanced pma4 promoters expressed GUS in the microspores and pollen grains ( Fig. 3M and P), thus retaining the tissue specificity of native pma4 ( Fig. 3K and N). But for the 35SGUS plants ( Fig. 3L and O), neither the microspores, nor the mature pollen grains displayed any GUS activity, as mentioned in previous reports [26,27]. ...
pma4 is the major plasma membrane H+-ATPase gene in Nicotiana plumbaginifolia. To study its physiological role by overexpression, we evaluated the possibility of enhancing the pma4 transcription promoter using a 165-bp enhancing sequence of the CaMV 35S transcription promoter. This was inserted into the pma4 promoter either 500 or 50 nucleotides upstream from the transcription start site. Transient expression with the gusA reporter gene showed that both enhanced pma4 promoters had a 4- to 13-fold greater transcription activity than the native pma4 promoter. Quantitative analysis of stable transgenic plants also showed that both enhanced pma4 promoters conferred much greater GUS activity. Histochemical assay showed that the enhanced promoters produced strong GUS activity in most cell types as already observed in the 35S or the native pma4 promoter. In the cell types where either the 35S or pma4 promoter was inactive, the enhanced promoters mimicked expression of the active one. However, there were cases (e.g. root cortex of seedlings) where, although both 35S and pma4 promoters were active, none of the enhanced promoters induced GUS activity. This might indicate the interference of promoter regulatory elements. The two enhanced pma4 promoters conferred similar expression throughout the plant development, implying that there was no regulatory element at either the pma4 −500 or −50 position, that conferred important tissue specificity.
Background
In many plants, the amino acid proline is strongly accumulated in pollen and disruption of proline synthesis caused abortion of microspore development in Arabidopsis. So far, it was unclear whether local biosynthesis or transport of proline determines the success of fertile pollen development.
Results
We analyzed the expression pattern of the proline biosynthetic genes PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 & 2 (P5CS1 & 2) in Arabidopsis anthers and both isoforms were strongly expressed in developing microspores and pollen grains but only inconsistently in surrounding sporophytic tissues. We introduced in a p5cs1/p5cs1 p5cs2/P5CS2 mutant background an additional copy of P5CS2 under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, the tapetum-specific LIPID TRANSFER PROTEIN 12 (Ltp12) promoter or the pollen-specific At5g17340 promoter to determine in which site proline biosynthesis can restore the fertility of proline-deficient microspores. The specificity of these promoters was confirmed by β-glucuronidase (GUS) analysis, and by direct proline measurement in pollen grains and stage-9/10 anthers. Expression of P5CS2 under control of the At5g17340 promoter fully rescued proline content and normal morphology and fertility of mutant pollen. In contrast, expression of P5CS2 driven by either the Ltp12 or CaMV35S promoter caused only partial restoration of pollen development with little effect on pollen fertility.
Conclusions
Overall, our results indicate that proline transport is not able to fulfill the demand of the cells of the male germ line. Pollen development and fertility depend on local proline biosynthesis during late stages of microspore development and in mature pollen grains.
Electronic supplementary material
The online version of this article (10.1186/s12870-018-1571-3) contains supplementary material, which is available to authorized users.
The development of highly sensitive assays for glycosidases is of critical significance to understand their functions, facilely detect associated diseases and screen potential new drugs. In this work, we develop a universal assay strategy for glycosidase enzymes and inhibitor screening based on functional carbon quantum dots through a combined host-guest recognition and specific static quenching-induced signal transduction mechanism. This detection strategy is established in terms of the following facts: (1) β-cyclodextrin as a perfect host can selectively associate with p-nitrophenol due to its hydrophobic character and right size match of the cavity, which renders specific binding between β-cyclodextrin and p-nitrophenol via a host-guest recognition. (2) The formation of an inclusion complex between β-cyclodextrin modified carbon quantum dots (β-CD-CQDs) and p-nitrophenol results in fluorescence quenching with a high quenching efficiency due to the static quenching mechanism. Glycoconjugates of p-nitrophenol as the substrates could be rapidly hydrolyzed to corresponding glycose and p-nitrophenol in the presence of specific glycosidase, and the resulting p-nitrophenol induces the following host-guest interaction and static quenching leading to a change in the fluorescence signal. The activity of different glycosidase enzymes could be evaluated in the same way as long as the glycosyl unit of glycosylated substrates was changed. Here we take β-galactosidase as an example to demonstrate the applicability of the proposed detection strategy because it can act as a molecular target for primary ovarian cancers. A highly sensitive assay for β-galactosidase activity in terms of linear correlation of the fluorescence change with the β-galactosidase level was established with a low detection limit of 0.6 U L⁻¹. Its function of inhibitor screening was also assessed by using d-galactal as the inhibitor for β-galactosidase, and the positive results indicated its feasibility to screen potential inhibitors. It is also illustrated that the nanoprobe possesses excellent biocompatibility, and can sensitively monitor the intracellular β-galactosidase level in ovarian cancer cells. This work provides a general detection method for glycosidase activity, demonstrates its applicability of monitoring the enzyme level in living cells, and broadens fluorogenic probes in fluorescence-guided diagnostics.
The first attempts to transform higher plants were published twenty-five years ago. Hess (1969) described with Petunia hybrida the partial correction of a white flowering mutant to anthocyanin synthesis type by incubating swelling and germinating seeds using wild type DNA; one year later, the transfer of a gene governing leaf shape was reported (Hess, 1970). Furthermore, in 1969 Ledoux and Huart reported on DNA uptake into Arabidopsis seedlings, and some years later on the DNA-mediated correction of thiamineless mutants of the same species (Ledoux, et al., 1974).
The development of pollen as a vector for direct gene transfer would be a significant advance in our ability to introduce genes into plants. Such methodology should be of general utility for many plant species, and in particular for the major monocotyledonous crop plants such as maize, wheat and barley that are recalcitrant to protoplast regeneration and that are not amenable to Agrobacterium based transformation techniques. A further advantage would be the avoidance of tissue culture steps that are time consuming and known to result in undesirable somaclonal variation. The potential of pollen as a vector for direct gene transfer has long been realized. For more than 10 years numerous investigators have attempted pollen-mediated transformation, several of which have claimed success [5, 8, 19, 23]. However the ultimate proof that transformation has taken place, that is, the demonstration of integration of foreign DNA into the nuclear genome at the molecular level and the genetic transmission of this DNA, is still lacking. This chapter presents a summary of research that has been directed towards pollen-mediated gene transfer, a detailed protocol for the delivery of DNA into pollen using particle bombardment and a discussion of factors that may be important for the successful application of this technique to obtain stably transformed plants.
For their reproductive success higher plants depend on the functionality and efficacy of a specialized structure, the flower. This contains specialized organs (anther and carpel) in which the male and female reproductive cells are produced and non-reproductive organs (sepal and petal) which attract pollinators and protect the developing seed. In the anther the microspores develop which after maturation become pollen grains. A pollen grain can be regarded as a free living haploid organism. It contains one vegetative and one generative cell in bicellular pollen or one vegetative cell and two sperm cells in tricellular pollen. Its main task is to deliver the two sperm nuclei through the female reproductive tissues to the egg cell. The pollen can be transported to the female organs by a variety of mediators such as wind or insect pollinators. After arrival on a compatible and receptive stigma the pollen grains germinate by the extrusion of a tube through a germ pore in the pollen wall. The pollen tube grows through the transmitting tissue of the style to reach the ovary. After the ovary has been entered, the tube grows through the micropyle of the ovule towards the embryo sac. The tube enters one of the synergids of the embryo sac and ruptures. The sperm cells are then released into the synergid. In a process known as double fertilization one sperm cell fuses with the egg cell to form the diploid zygote while the other sperm cell fuses with the central cell to form the triploid endosperm. For most angiosperms, pollen germination, tube growth and fertilization are rapid events which are completed within 1 to 48 hours. At the morphological level a wealth of knowledge about the formation of the reproductive organs and cells, pollen germination, and the fertilization process has been gained during the last 50–100 years. This is in sharp contrast with the availability of data describing molecular processes regulating the different steps of plant reproduction. Only limited data exist describing the role of gene products or signal molecules which interact in and control the development of male and female reproductive cells and the fertilization process.
One of the major reasons leading to the popularity of the beta-glucuronidase (GUS)-fusion system (1) was the general assumption that there was no detectable intrinsic GUS activity in higher plants (2). Nevertheless, more and more investigators were troubled by the so-called “false positive” results or “background” activities when GUS assays were performed. We decided to look into this phenomenon with a large variety of selected seed plants. Positive GUS tests were observed in certain parts of some Gymnosperm and every key group of Angiosperm species. In fact, the presence of GUS activities in seed plants were documented in a handful of early reports (reviewed in 3) and some recent reports (4,5).
The control of gametophytic self-incompatibility as exhibited in most plant species is exerted through the action of a single locus S. When haploid pollen grains genotype Sx alight on a style expressing the same S allele, fertilization is prevented, whereas pollen grains whose S-alleles are not matched by those expressed in the style grow, and potentially effect fertilization (de Nettancourt, 1977). The mechanism by which pollen cells are distinguished on the basis of their S-alleles has been studied by physical, biochemical and more recently molecular techniques, without a complete picture having so far emerged. The best-studied system is that of the Solanaceae and this will form the focus of this paper.
We have evaluated T-DNA mediated plant promoter tagging, with a left-border-linked promoterless firefly luciferase (luc) construct, as a strategy for the isolation of novel plant promoters. In a population of approximately 300 transformed tobacco plants, IO lines showed LUC activity, including novel tissue-specific and developmental patterns of expression. One line, showing LUC activity only in the shoot and root apical meristems, was further characterised. Inverse PCR was used to amplify a 1.5 kb fragment of plant DNA flanking the single-copy T-DNA insertion in this line. With the exception of a 249 bp highly repetitive element, this sequence is present as a single copy in the tobacco genome, and is not homologous to any previously characterised DNA sequences. Sequence analysis revealed the presence of several motifs that may be involved in transcriptional regulation. Transgenic tobacco plants transformed with a transcriptional fusion of this putative promoter sequence to the beta-glucuronidase (uidA) reporter gene, showed GUS activity confined to the shoot tip and mature pollen. This promoter may be useful to direct the expression of genes controlling the transition to flowering, or genes to reduce losses due to pests and stresses damaging plant apical meristems.
As an alternative to the traditional histochemical GUS visualization substrate, 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), the fluorescence substrates ImaGene Green™ (C12FDGLcU) and ImaGene Red™ (C12RGlcU) were used to visualize GUS activity in transformed cassava cells. These fatty acyl-containing GUS substrates become anchored to the cell membrane, by means of hydrophobic interactions with the plasma membrane. Once anchored to the cell membrane, the fluorogenic glucosidic moiety is transferred to the cytosolic side of the membrane, thereby introducing the fluorogenic substrate into the cell without the need to solubilize the cell membranes. Using these two fluorogenic and lipophilic substrates, many of the practical difficulties often experienced in the visualization of GUS activity in transformed cells with X-Gluc can be readily overcome.
Accurate detection of a genetic or biochemical marker introduced into sugarbeet (Beta vulgarjs L.) is based on the absence of native sequences or activities in the plant that could confound the analysis of expression of the introduced marke r. During the course of experiments designed to optimize DNA transfer from Agrobacterjum tumef aciens to sugarbeet leaf disc cells, an endogenous enzyme activity was discovered which utilizes all the common substrates recognized by the marker enzyme l3,-glucuronidase (GUS) from E. coli. This native sugarbeet enzyme (SB-GUS) was characterized immunologically and biochemically. GUS and SB-GUS were found to be distinct with regard to pH optima, thermal inactivation, reaction to denaturants and protein modifying reagents, inhibition by metals and saccharo-Iactone, and molecular mass. The two activities are not immunologically related, as judged by Western blot and immunoprecipitation analyses. A protocol was developed to accurately quantitate introduced GUS in the presence of SB-GUS, by utilizing selective inhibition of GUS at pH 7.0 by saccharic acid l,4-lactone. Under these conditions GUS activity is completely eliminated, while SB GUS activity was unaffected.
Sedentary endoparasitic nematodes induce multinucleate feeding cells in the roots of their host plants. These cells undergo multiple rounds of shortened cell cycles leading to genome amplification and hypertrophy of the cytoplasm. After explaining the specific terminology involved, this chapter reviews the cytological observations in giant cells induced by root-knot nematodes and syncytia induced by cyst nematodes. Recent molecular research into cell cycle regulation in Arabidopsis thaliana has been extended from normal plant development to the changes induced by these sedentary nematodes. In this way it has been demonstrated that several cell cycle genes are activated in developing feeding cells. To analyze the importance of cell cycle activation for the establishment of feeding cells, cell cycle-inhibiting drugs have been used and experiments with plants producing dominant mutant proteins will be performed in the near future.
Compared to bacterial and fungal phytopathology, and in contrast to semiochemical research related to insect behavior, the knowledge of plant—nematode interactions and the chemical signaling involved is limited and progressing slowly. Their complex life cycles and the small size of these obligate parasites are major stumbling blocks for experimental approaches.
Today a number of cell culture and molecular techniques are used or are under development to manipulate sexual reproduction. Protoplast fusion is used not only to create somatic hybrids but also to produce cybrids that regenerate into cytoplasmic male sterile plants. Unreduced gametophytes are selected for polyploidization or distant hybridization. In vitro pollination and in vitro fertilization after isolation of egg and sperm cells are used to overcome pre- and postfertilization crossing barriers (Shivanna and Johri, 1985).
Introduction Mechanisms of Engineering Glyphosate Resistance Development of First-Generation GR Crops Identification of at-Risk Tissues for Glyphosate Injury Development of Second-Generation GR Crops Use of Alternative Herbicides for Weed Resistance Management Engineering Crop Resistance to the Glufosinate Herbicide Engineering Crop Resistance to the Dicamba Herbicide Stacking of Herbicide-Resistant Traits Acknowledgments References
We have analyzed the spatial and temporal activities of chalcone flavanone isomerase (chi) A and B gene promoters from petunia. To study the tandem promoter regulation of chiA, various chiA promoter fragments were fused with the beta-glucuronidase (GUS) reporter gene. Analysis of transgenic plants containing these chimeric genes provided definitive proof that the chiA coding region is regulated by two distinct promoters (designated PA1 and PA2). We also showed that both promoters can function independently and that the chiA PA1 promoter is expressed in limb (epidermal and parenchyma cells), tube (inner epidermal and parenchyma cells), seed (seed coat, endosperm, and embryo), sepal, leaf, and stem. The use of chiA and chiB promoters in the regulation of anther- and pollen-specific gene expression has been studied. By analyzing transgenic plants containing chimeric genes consisting of chiA and B promoter fragments and the GUS reporter gene, we were able to identify a 0.44-kilobase chiA PA2 promoter fragment that drives pollen-specific gene expression and a 1.75-kilobase chiB PB promoter fragment that confers anther-specific (pollen and tapetum cells) expression to the GUS gene.
The emergence of synthetic glycoconjugates as chemical probes for the detection of glycosidase enzymes has resulted in the development of useful chemical tools with applications in glycobiology, biotechnology, medical and industrial research. Critical to the function of these probes is the preparation of substrates containing a glycosidic linkage that when activated by a specific enzyme or group of enzymes, irreversibly releases a reporter molecule that can be detected. Starting from the earliest examples of colorimetric probes, increasingly sensitive and sophisticated substrates have been reported. In this review we present an overview of the recent advances in this field, covering an array of strategies including chromogenic and fluorogenic substrates, lanthanide complexes, gels and nanoparticles. The applications of these substrates for the detection of various glycosidases and the scope and limitations for each approach are discussed.
Transfer of the GUS gene into pollen has been studied in co-cultures of Agrobacterium and Petunia pollen. The Agrobacterium strain used contains a GUS gene between the two borders of the T-DNA. Uptake and integration of this GUS gene could be shown using two different restriction systems. First, by appropriate cleavage within the T-DNA the GUS gene could be isolated intact from Agrobacterium-treated pollen. Second, using enzymes with cleave the T-DNA only once, integration of this T-DNA into individual pollen genomes could be shown. The fragments obtained could not be obtained from Agrobacterium alone. The positive Southern blots were reprobed with vir probes, but all were negative. Also following plating, no Agrobacteria could be detected from our pollen DNA preparations. Therefore, the signals obtained were not due to contaminating bacteria. Due to a high endogenous GUS activity of Petunia pollen the expression of the transferred gene could not be studied. The data demonstrate the uptake and integration of T-DNA into pollen and are closing a gap in the line of evidence for the functioning of an indirect Agrobacterium — mediated gene transfer. Besides this, it should be stressed that only this indirect pollen system leads to success.
We optimized the biological and physical parameters for DNA delivery into thalli of the red alga Porphyra yezoensis using a particle bombardment device. The efficiency of transformation was determined using the {\beta}-glucuronidase (GUS) assay. The optimal helium pressure, distance of tungsten particle flight, and ratio of DNA to tungsten particles were 23kgf/cm^2, 8 cm, and 5?g/mg tungsten, respectively. During bombardment, osmotic treatment with a mixture of 0.6 M mannitol and sorbitol increased the efficiency of GUS transformation. After 2 days, the blue color indicating GUS activity was observed using a histochemical assay.
As part of our research to develop an alternative system for the transformation of recalcitrant plant species we investigated the use of the male gametophyte as a transformation vector. Therefore the activity of four different promoters (CaMV 35S, LAT52, chiA PA2 and TR2') was analyzed in pollen of a dicot (Nicotiana glutinosa) and a monocot (Lilium longiflorum) plant species. Gene constructs in which the ß-glucuronidase (GUS) gene was placed under the control of these promoters were introduced in pollen using a particle delivery system. No activity of the Cauliflower Mosaic Virus (CaMV) 35S promoter was detected in pollen of both N. glutinosa and L. longiflorum. The promoter of the tomato flower-specific LAT52 gene was highly active in N. glutinosa pollen but remained silent in L. longiflorum pollen. A similar expression pattern was observed for the pollen-specific Chalcone Flavanone Isomerase chiA PA2 promoter originally isolated from petunia. The TR2' mannopine synthase promoter of Agrobacterium tumefaciens, however, was active in pollen from Solanaceous species and also in pollen from the monocot L. longiflorum. This suggests that the TR2' promoter is active in vegetative and sporogenous tissues of dicot and monocot plant species.
Expression of marker genes, neomycin phosphotransferase (NPT) II and ß-glucuronidase (GUS), in the R1 progeny originating from a transgenic mustard plant line was investigated. The R1 shoots grown in the absence of kanamycin for 6 months showed reduced GUS activity in various tissues and an inability to form roots in the presence of kanamycin. Protoplasts isolated from these shoots expressed GUS at a frequency of 1-5% after 3, 10, 14 and 28d of culture, whereas wild type protoplasts did not show GUS activity throughout the process of somatic embryogenesis. Exogenous application of 20 μM 5-aza-cytidine greatly enhanced the frequency of GUS expressing cells, ranging from 26 to 34%, but not for the wild type during somatic embryogenesis. In protoplast-derived plants, GUS activity was detected in 4 out of 9 plants analysed. Southern analysis revealed that the NPTII gene was present in all GUS positive plants, but absent in GUS negative plants. The pattern of GUS expression in leaves was age-dependent irrespective of the plant developmental stage. GUS was strongly expressed in mature leaves, which was concomitant with an accumulation of a 1.8 kb GUS transcript. In contrast, GUS activity was either absent or occurred only at serrated tips of the young leaves, in which the GUS transcript levels were barely detectable. This study shows transgene instability in R1 mustard plants, which may be due to DNA methylation and/or deletion of the transgenes.
Global warming has seriously decreased world crop yield. High temperatures affect development, growth, and particularly, reproductive tissues in plants. A gene encoding β-ureidopropionase (SlUPB1, EC 3.5.1.6) was isolated from the stamens of a heat tolerant tomato (CL5915) using suppression subtractive hybridization. SlUPB1 catalyzes the production of β-alanine, the only beta-form amino acid in nature. In the anthesis stage, SlUPB1 expression in CL5915 stamens, growing in 35/30°C (day/night), was 2.16 and 2.93 times greater than that in a heat-sensitive tomato (L4783) cultivated in 30/25°C or 25/20°C, respectively. Transgenic tomatoes, upregulating SlUPB1 in L4783 and downregulating SlUPB1 in CL5915, were constructed, and the amount of β-alanine measured by LC-ESI-MS in the transgenic overexpression of SlUBP1 was higher than that of L4783. However, the β-alanine in the transgenics downregulating SlUBP1 was significantly lower than the β-alanine of CL5915. Pollen germination rates of these transgenics were analyzed under different developmental and germinating temperatures. The results indicated that germination rates of transgenics overexpressing SlUBP1 were higher than germination rates of the background tomato L4783. Germination rates of transgenics downregulating SlUPB1 were significantly lower than germination rates of background tomato CL5915, indicating the necessity of functional SlUPB1 for pollen germination. Pollen germinating in the buffer with the addition of β-alanine further indicated that β-alanine effectively enhanced pollen germination in tomatoes with low SlUPB1 expression. Together these results showed that the expression of SlUPB1 is important for pollen germination, and β-alanine may play a role in pollen germination under both optimal and high temperatures.
This chapter discusses the reporter genes. Differential gene expression is essential for the development of higher organisms. Transcriptional regulation appears to play a key role in this process. However, the relevant gene products may be difficult to detect due to low abundance or chemical properties. Therefore, the analysis of developmental processes is greatly simplified if promoters or other regulatory sequences are linked to reporter genes. Likewise, reporter enzymes that remain active when fused to other peptides allow the study of protein trafficking in the cell. To compare enzyme activities, standardization of extracts is necessary. The most convenient method is determining the protein content of extracts—for example, using the assay described by Bradford. Enzyme kinetics are characterized by a lag, linear, and stationary phase. During the linear range, substrate accumulation is proportional to enzyme concentration. Enzyme assays are quantitative only within the linear phase, which may have to be determined initially. The frequent use of GUS is mainly justified because it allows the histochemical localization of enzyme activity in tissues or complete plantlets. Also, after the initial investment in a fluorimeter, a fluorogenic assay provides a relatively inexpensive and highly sensitive method to quantify GUS activity.
烟草绒毡层特异启动子 pTA29 (TA29 promoter)已用于多种植物雄性不育和花粉发育的研究。作为外源特异表达启动子 pTA29 在棉花中的表达特性尚不清楚，为了研究该启动子在棉花中的表达特异性，本文构建了 pTA29:gus 融合基因，并将其转入棉花。研究发现在GUS染液中添加10%的乙醇可以抑制棉花花药内源GUS活性。用改良的GUS染液进行组织化学染色表明， pTA29:gus 转化植株的GUS活性主要存在于花药中，并在花蕾长为6 mm和15 mm的花药中有2个活性高峰，而在根、茎、胚珠、花瓣、苞叶中未被检测到。与烟草不同，在 pTA29:gus 棉花转化植株的叶片表皮毛和花粉中也能检测到GUS活性。定量RT-PCR分析表明转化子中gus基因的转录水平与GUS活性一致。上述结果表明，烟草绒毡层特异启动子 pTA29 可以控制下游基因在棉花花药中优势表达；在利用该启动子进行棉花基因功能以及雄性不育研究时，应注意该启动子在棉花中的表达范围和组织特异性。
A conspicuous endogenous maize (Zea mays L.) β‐glucuronidase (GUS) activity was observed in histochemical assays of non‐transformed maize kernels, confounding the use of Escherichia coli gusA as a reporter gene. Appearance of the endogenous activity was developmentally dependent and highly tissue‐specific, being localized to the upper pedicel (basal maternal kernel) tissues where the black layer forms in the latter stages of kernel development. Pedicel homogenates exhibited GUS activity using either p‐nitrophenyl‐β‐D‐glucuronide or 4‐methylumbelliferyl‐β‐D‐glucuronide (MUG) as substrates. Pedicel GUS was apparently not the result of endophyte contamination of enzyme isolates since no endophytes could be cultured. The MUG‐based activity had a pH optimum of 4 to 5 and was separable into two isoforms by anion exchange chromatography with Km values for MUG of 2.2 and 2.7 µM for the early‐ and late‐eluting forms, respectively. The pedicel GUS isoforms had very similar characteristics: native Mr of approximately 32000, stimulation by assay at 60°C, inhibition at high ionic strength or in the presence of EDTA and relative insensitivity to the E. coli GUS inhibitor saccharic acid‐1,4‐lactone. Only the early‐eluting form, however, was capable of hydrolyzing the histocbemical GUS substrate 5‐bromo‐4‐chloro‐3‐indoyl‐β‐D‐glucuronide. Neither isoform exhibited antifungal activity against Fusarium moniliforme. In contrast to the in vitro activity, pedicel endogenous GUS measured histochemically was completely inhibited by saccharic acid‐1,4‐lactone, unaffected by EDTA and greatly decreased by incubation at elevated assay temperature. A modification of the standard histochemical GUS assay allowed complete suppression of endogenous GUS activity while enhancing E. coli‐derived GUS activity in kernels transiently expressing the gusA gene. Possible roles of these endogenous GUS activities within the black layer region of the kernel pedicel are proposed.
Scutella of immature barley embryos were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into the scutella by biolistic transformation. The amount of intrinsic activity was strongly genotype dependent and could be detected with both the histochemical and fluorimetrical assay. This interfering activity could not be eliminated by performing the assay at pH 7.0 nor using additional methanol in the incubation buffer. Only pretreatment of the bombarded scutella for 60 min at 55°C followed by staining at the same temperature in a modified buffer was found to completely suppress barley-endogenous β-glucuronidase. In addition to the spots of strong GUS activity, these improved conditions made it possible to observe high numbers of smaller GUS-spots that became visible on the white scutellu, being no longer masked by a previous non-specific blue staining pattern. Further, this protocol can be used for unequivocally differentiating between transgenic and non-transgenic barley plants.
One of the problems associated with the use of the bacterial gus A gene as a reporter is the presence of ‘endogenous GUS’ activity in some plant species. A recent paper [1] suggests that the addition of methanol to GUS reaction buffer specifically inhibits endogenous GUS activity while enhancing the activity of an introduced gus A gene. The results described in this paper suggest this phenomenon may be explained by the effect methanol has on cell membranes and demonstrate that it does not specifically inhibit endogenous GUS activity.
Bacterial β-glucuronidase is often introduced into plants as a reporter gene fused to constitutive or inducible promoters. However, the presence of both endogenous inhibitors of GUS activity and endogenous GUS enzymes in transgenic plants could lead to an underestimation of GUS. In this paper, a decrease of the Vm values and a greater affinity (Km) of the GUS enzyme for its substrate (p-NPG) has been recorded when increasing amounts of protein from untransformed tobacco cells has been added to the pure β-glucuronidase. The observed inhibition is not competitive and can be completely removed when the tobacco extracts are passed through Sephadex G-25 spin columns prior to the assays. After such a treatment, the activity of E. coli GUS in transgenic tobacco cells (constitutive or inducible systems) was stimulated by a factor 1.2 or 2 for p-NPG or 4-MUG substrates, respectively. This method was also effective in suppressing the endogenous GUS or GUS-like activity which can interfere with the activity originating from the introduced GUS gene.
We report the transfer of kanamycin resistance of bacterial origin into wheat via pipetting Agrobacterium suspension into wheat spikelets. Grain progenies obtained were screened for kanamycin resistance in a two-step procedure allowing the elimination of infected grains. As shown by Southern blot analysis and NPTII-activity assays the gene conferring kanamycin resistance had been transferred into the first and second sexual generation. The transformants obtained were fully fertile and showed no morphological abnormalities. Taking all experiments together, including some unsuccessful ones, transformation frequency was 1%, the highest transformation frequency in a single experiment being 2.6%. All transformants showed alterations concerning the transferred DNA sequences. In the selfed progenies of some first generation transformants the foreign gene had been lost. NPTII-activity was compartively low. Several tests including probing with vir probes prove that our transformants are not contaminated with Agrobacterium. Using the model plant Petunia hybrida, Agrobacterium-mediated gene transfer via pollen had been proven previously. As Petunia pollen, wheat pollen contain diffusible factors identified as flavonol glycosides which induce the plasmidial vir region. By pipetting Agrobacterium suspension into wheat spikelets, bacteria and pollen are brought in intimate contact. Therefore, pollen-mediated gene transfer seems the best explanation for the results obtained. The (pollen) system used in our experiments could be easily adapted to other plant species. It represents an alternative to protoplast-based transformation and leaf disk techniques by avoiding regeneration problems and somaclonal variation, introducing fertile plants. Our procedure is further characterized by its high efficiency and simplicity.
Intravital observations of pollen grains and pollen tubes of 34 plant species with particular consideration of spherosomes have been carried out. In a part of the material the localization of the following hydrolases was investigated: acid and alkaline phosphatase, non-specific esterase, lipase, deoxyribonuclease, β-gluouronidase and arylsulphatase. The results indicate that these hydrolases are present in pollen 'grains and tubes of all the plant species investigated. The only exception is alkaline phosphatase, its occurrence not being general. The activity of acid phosphatase, non-specific esterase, arylsulphatase, β-glucuronidase and probably deoxyribonuclease is localized in the spherosomes of pollen grains and tubes. The localization of alkaline phosphatase has not been exactly established. The author did not observe penetration of acid phosphatase into the environment of the tube as described in the literature. Spherosomes occurring in agglomerates - elaioplasts - appear to have the same morphologic features and optical properties and the same set of hydrolytic enzymes. The top zone of young, growing pollen tubes is devoid of spherosomes. Cytoenzymic investigations have shown no presence of acid phosphatase in this zone.
Effects of kanamycin on pollen germination and tube growth of pollen from non-transformed plants and from transgenic tomato plants containing a chimaeric kanamycin resistance gene were determined. Germination of pollen was not affected by the addition of kanamycin to the medium in both genotypes. Kanamycin, however, severely affected tube growth of pollen from non-transformed plants, while pollen from plants containing the chimaeric gene were less sensitive and produced significantly longer tubes at kanamycin concentrations between 200-400 mgl-1. Apparently, this resistance for kanamycin correlates with the expression of the chimaeric gene during male gametophytic development.
Tuber discs of Solanum tuberosum cv Bintje and Désirée were cocultivated with an Agrobacterium tumefaciens binary vector, carrying both the neomycine phosphotransferase and the E. coli β-glucuronidase gene fused to resp. the nopaline synthase and Cauliflower mosaic virus 35S promotor.
Inoculated tuber discs produce transgenic shoots in selective media containing kanamycin. The transgenic plants are phenotypically normal and contain the euploid number of chromosomes. Both the neomycin phosphotransferase as well as the β-glucuronidase gene are expressed conferring resp. kanamycin resistance and β-glucuronidase activity to the plants.
The synthesis of β-galactosidase during Brassica campestris pollen development results from the transcription of the haploid genome. A quantitative cytochemical method has been developed in which 5-bromo-4-chloro-3-indoxyl-β-d-galactoside is used as substrate giving a blue-green final reaction product. We have recently detected oilseed rape plants which are heterozygous for the β-galactosidase locus, in which 50% of the pollen grains produced are Gal (having enzyme activity), while the other 50% are gal (enzyme deficient). The gal pollen grains served as a built-in control during microspectrophotometric determinations of enzyme activity. The present study has identified the developmental phase at which synthesis of the enzyme commenced. Activity is absent in microsporocytes, tetrads, and at microspore release. Enzyme activity is first detected in the young microspores and, by early vacuolate period, there is an increase in the rate of enzyme activity. A second period of increased enzyme synthesis occurred prior to generative cell division, although the rate is reduced in mature pollen.
The synthesis of beta-galactosidase during Brassica campestris pollen development results from the transcription of the haploid genome. A quantitative cytochemical method has been developed in which 5-bromo-4-chloro-3-indoxyl-beta-d-galactoside is used as substrate giving a blue-green final reaction product. We have recently detected oilseed rape plants which are heterozygous for the beta-galactosidase locus, in which 50% of the pollen grains produced are Gal (having enzyme activity), while the other 50% are gal (enzyme deficient). The gal pollen grains served as a built-in control during microspectrophotometric determinations of enzyme activity. The present study has identified the developmental phase at which synthesis of the enzyme commenced. Activity is absent in microsporocytes, tetrads, and at microspore release. Enzyme activity is first detected in the young microspores and, by early vacuolate period, there is an increase in the rate of enzyme activity. A second period of increased enzyme synthesis occurred prior to generative cell division, although the rate is reduced in mature pollen.
During male gametophyte development the synthesis of several proteins occurs from transcripts of the haploid genome. Alcohol dehydrogenase (ADH1), a developmentally regulated protein, was chosen for study to determine the stage at which its synthesis was initiated and the pattern of its synthesis during microsporogenesis. The ability of ADH to reduce p-nitro blue tetrazolium chloride in situ was used as an indicator of enzyme activity. Maize strains heterozygous for adh1 were utilized to provide an internal control, 50% of the grains being adh1(+) and 50% being adh1(-). No ADH activity was detectable when tetrads were first formed after meiosis. Activity was initially detected soon after the tetrads began to break apart but before the microspores in the tetrads had completely separated. The transcription of the adh1 gene from the haploid genome must thus occur very soon after meiosis is completed. ADH activity increases at a constant rate thereafter until microspore mitosis when an increase in the rate takes place which lasts until generative cell division. Thereafter, there is a marked decrease in the rate of accumulation of ADH activity.
Brewbaker, James L., and Beyoung H. Kwack. (U. Hawaii, Honolulu.) The essential role of calcium ion in pollen germination and pollen tube growth. Amer. Jour. Bot. 50(9): 859–865. Illus. 1963.—A pollen population effect occurs whenever pollen grains are grown in vitro. Small pollen populations germinate and grow poorly if at all, under conditions which support excellent growth of large pollen populations. The pollen population effect is overcome completely by a growth factor obtained in water extracts of many plant tissues. This factor is shown to be the calcium ion, and its action confirmed in 86 species representing 39 plant families. Other ions (K⁺, Mg⁺⁺, Na⁺) serve in supporting roles to the uptake or binding of calcium. The high requirement of calcium (300–5000 ppm, as Ca (NO3)2·4H2O, for optimum growth) and low calcium content of most pollen may conspire to give calcium a governing role in the growth of pollen tubes both in vitro and in situ. It is suspected that ramifications of this role extend to the self-incompatibilities of plants and to the curious types of arrested tube growth distinguishing, for example, the orchids. A culture medium which proved its merit in a wide variety of pollen growth studies included, in distilled water, 10% sucrose, 100 ppm H3BO3, 300 ppm Ca (NO3)2·4H2O, 200 ppm MgSO4·7H2O and 100 ppm KNO3.
The mechanism by which any organism controls the production of proteins by the differential expression of its genes has fascinated many researchers since protein production and gene expression were first linked. It has been shown that in most cases the production of polypeptides is mainly controlled at the level of gene transcription, although in some cases mRNA or protein stability might also play an important role. In many instances the detection of a polypeptide of interest, that may only be produced under certain conditions or in specific cell types within an organism, is rather complex or laborious since many of these gene products do not have easily detectable enzymatic or functional activities.
High resolution cytochemical methods have been used to characterize pollen development and pollen-wall structure in Helianthus annuus. Aniline-blue fluorescent material, presumably callose, was detected in the nexine layer throughout its development. It was associated with acid phosphatase activity, while the outer sexine possessed intense esterase activity during the young spore period. Acid phosphatase and esterase were present in both intine and exine wall sites in contrast to their specific location in other pollen types. Quantitative cytochemical estimates of enzyme activity during development reveal esterase patterns typical of gametophytic synthesis, while acid phosphatase patterns are characteristic of sporophytic origin suggesting tapetal transfer during the vacuolate period.
Pollination is a process that is vital for the survival not only of flowering plants, but also for mankind. Yet human efforts to manipulate the reproduction of crop plants have been largely empirical. Today, research into plant reproduction is not given high priority. Yet it provides the means of ensuring successful pollination, in order to maximize fertilization and hence crop production. The pollination process is susceptible to modification to permit the introduction of desirable genes, for example, for disease resistance, from wild species into crop plants. The potential applications are endless, yet all depend on a precise knowledge of the events of pollination (see reviews by STanley and LInskens 1974, FRänkel and GAlun 1977, CLarke and KNox 1978).
Under humid conditions, both bi- and trinucleate pollen species incorporate, on the average, very low amounts of leucine, e.g., 0.4 pmol min(-1)mg pollen(-1). During germination in vitro, however, the two types of pollens greatly differ in their capacity for protein synthesis.Binucleate pollen species such as Typha, which are characterized by slow respiration in humid air and prolonged lag periods during germination in vitro, contain large amounts of monoribosomes at dehiscence. Polyribosomes are formed soon after the pollen is wetted in the germination medium, and a considerable incorporation of leucine is initiated after 10-15 min. More rapidly respiring, binucleate pollen showing a short lag period, such as Tradescantia, may already contain many polysomes at dehiscence and incorporate leucine within 2 min of germination. However, rapidly respiring, trinucleate Compositae pollen contains very limited amounts of ribosomal material and never attains any substantial level of incorporation. Cycloheximide completely inhibits both protein synthesis and tube emergence and growth in the slowly respiring, binucleate pollen species. The more rapidly respiring types are less dependent on protein synthesis, while germination of the phylogenetically advanced, trinucleate Compositae pollen proceeds completely independently. It is concluded that the level of phylogenetic advancement of the male gametophyte is characterized by its overall state of metabolic development at dehiscence rather than by the number of its generative cells.
The purpose of this paper was to determine if heavy metal tolerance was expressed in pollen and if its expression was correlated with the tolerance of the pollen source. Clones of Silene dioica, tolerant to zinc, closely related but nontolerant S. alba and clones of Mimulus guttatus tolerant and sensitive to copper were grown in the greenhouse in either standard potting soil or nutrient culture. Pollen was collected shortly after dehiscence, hydrated, and tested over a broad range of metal concentrations. The tolerance of the pollen source was determined by comparing root growth in solutions with and without heavy metals. In both Silene species and M. guttatus, the tolerance of the parental clone was expressed in its pollen. Pollen from tolerant individuals was able to germinate and grow at concentrations of metals which markedly inhibited pollen from nontolerants.
To determine the extent of gametophytic gene expression and the type of transcription, haploid or haplo-diploid, of the genes, isozymes were used as genetic markers. Fifteen enzymatic systems, including thirty-four isozymes, were studied. The determination of the type of expression of genes coding for multimeric enzymes was based on the comparison of electrophoretic patterns of pollen and of sporophytic tissues from plants heterozygous for electrophoretic mobility: if gene expression in pollen is of a gametophytic (haploid) origin, pollen, unlike the sporophyte, would reveal only the parental homomultimeric bands. The enzymes analyzed can be grouped in three categories according to type of gene expression: i) enzymes present in both pollen and sporophyte, coded by the same gene with haplo-diploid expression; ii) enzymes controlling analogous functions in pollen and sporophyte, coded by different genes, expressed in only one of the two phases; iii) enzymes present in two or more forms in the sporophyte and only in one form in the gametophyte. The data allow the proportion of haplo-diploid gene expression in the loci examined to be estimated at 0.72; 0.22 and 0.06 being the proportions attributable to the sporophytic and gametophytic domains, respectively.
The reporter genes for Chloramphenicolacetyltransferase (CAT), Neomycinphosphotransferase-(NPT)-II and β-Glucuronidase (GUS) were compared in transient gene expression experiments in tobacco mesophyll protoplasts. For this purpose, nearly identical chimeric genes controlled by the CaMV 35 S promoter were constructed. The detection level of each system was determined yielding the following order of relative sensitivity: CAT<NPT II<GUS.
Tomato genotypes superior in regenerating plants from protoplast and callus cultures were obtained by transferring regeneration capacity from Lycopersicon peruvianum into L. esculentum by classical breeding. The genetics of regeneration and callus growth have been studied in selfed and backcross progenies of a selected plant (MsK93) which has 25% L. peruvianum in its ancestry. Segregation data showed that the favourable cell culture traits of L. peruvianum are dominant. Regeneration capacity from established callus cultures was controlled by two dominant genes. Callus growth on primary expiants, callus growth of established cultures and shoot regeneration from explants had high heritabilities (0.47, 0.78, 0.87, respectively). Callus growth and regeneration capacity were not correlated within the populations studied.
Effects of the phytotoxic compounds (AAL-toxins) isolated from cell-free culture filtrates of Alternaria alternata f.sp. lycopersici on in vitro pollen development were studied. AAL-toxins inhibited both germination and tube growth of pollen from several Lycopersicon genotypes. Pollen from susceptible genotypes, however, was more sensitive for AAL-toxins than pollen from resistant plants, while pollen of species not belonging to the host range of the fungus was not significantly affected by the tested toxin concentrations. AAL-toxins elicit symptoms in detached leaf bioassays indistinguishable from those observed on leaves of fungal infected tomato plants, and toxins play a major role in the pathogenesis. Apparently, pathogenesis-related processes and mechanisms involved in disease resistance are expressed in both vegetative and generative tissues. This overlap in gene expression between the sporophytic and gametophytic level of a plant may be advantageously utilized in plant breeding programmes. Pollen may be used to distinguish susceptible and resistant plants and to select for resistances and tolerances against phytotoxins and other selective agents.
The contribution deals with the distribution of β-glucuronidase in the germinating pollen grains ofPortulaca grandiflora. In non-germinating pollen grains the enzyme is localized in the pollen wall; the cytoplasmic activity is subdued. With the
initiation of germination, the activity of enzyme increases and the positive granules are richly packed in the pollen grains
and pollen tubes. The stigma hairs also have such an activity. The functions of the enzyme in the metabolism of germinating
pollen grains are discussed.
Transformed petunia, tobacco, and tomato plants have been produced by means of a novel leaf disk transformation-regeneration
method. Surface-sterilized leaf disks were inoculated with an Agrobacterium tumefaciens strain containing a modified tumor-inducing plasmid (in which the phytohormone biosynthetic genes from transferred DNA had
been deleted and replaced with a chimeric gene for kanamycin resistance) and cultured for 2 days. The leaf disks were then
transferred to selective medium containing kanamycin. Shoot regeneration occurred within 2 to 4 weeks, and transformants were
confirmed by their ability to form roots in medium containing kanamycin. This method for producing transformed plants combines
gene transfer, plant regeneration, and effective selection for transformants into a single process and should be applicable
to plant species that can be infected by Agrobacterium and regenerated from leaf explants.
The efficiency of direct gene transfer to plant protoplasts has been increased more than 1000 fold over that previously reported. On the order of 2 percent of all colonies recovered without selection were transformed. The improvement was due to: treatment of protoplasts with a high voltage electric pulse (electroporation), optimization of the polyethylene glycol (PEG) concentration, addition of PEG after the DNA and the application of a heat shock (5 minutes at 45°C). These factors are of equal importance in achieving high transformation efficiencies.
The mRNAs of the mature pollen grain of Zea mays and of vegetative shoots were analyzed by comparing the kinetics of hydridization between homologous and heterologous reactions of cDNA to poly-(A)RNA in excess. The total complexity of pollen poly-(A)RNA i s 2.4107 nucleotides, whereas in the seedling shoot it is 4.0107 nucleotides. This corresponds to 24,000 different sequences in pollen and about 31,000 diverse mRNAs in shoots. The mRNAs in pollen can be divided into three abundance classes that constitute 35%, 49% and 15% of the total mRNAs having complexities of 2.1105, 6.4106 and 1.8107 nucleotides, respectively. Estimates have been made of the number of copies of each sequence per pollen grain or shoot cell, and also of the pollen mRNA sequences shared with those of the shoot.
The pea rbcS-3A gene codes for the small subunit of ribulose-1,5-bisphosphate carboxylase and its expression is regulated by light, tissue type and stage of development. Analysis in transgenic tobacco plants has shown that the upstream region contains an enhancer-like element which can confer light-regulated and organ-specific expression upon a reporter gene (Fluhr et al. 1986a). Here we address the question of whether the enhancer specifies not only organ specificity, but also expression in the correct cell types. In situ immunofluorescence and microdissection were used on transgenic tobacco plants containing the rbcS-3A enhancer fused to a reporter gene consisting of the cauliflower mosaic virus 35S promoter and the bacterial gene encoding chloramphenicol acetyltransferase (CAT). CAT levels are high in leaf mesophyll cells, but in the epidermis expression is restricted to guard cells. In the midrib, of the leaf and in the stem, there is considerable signal in the chlorenchyma and in the phloem region. This pattern of expression closely correlates with the distribution of the endogenous RbcS polypeptides and with the presence of chlorophyll. Our results demonstrate that the rbcS-3A enhancer-like element possesses all the necessary DNA sequences for expression in the correct cell types.
Mutants of E. coli K 12 unable to grow on a -D-glucuronide (methyl--D-glucuronide) and yet able to grow on hexuronates have been isolated; they are deficient for the enzyme: -D-glucuronidase. By conjugation and transduction experiments, these mutations have been mapped in a locus uid A between man and aro D. No or weak cotransduction is found between aro and man: man is shifted from its precedent location to 30.5 minute very close to uid A (linked by 83 to 97% of cotransduction). The order of loci in this region appears to be: ... phe S...pps...aro D...uid A...man.... Some of these mutations can reverse to wild type with a thermosensitive glucuronidase. These reverse mutations are found linked to this locus uid A; these results strongly suggest that uid A is the site of the structural gene of -glucuronidase.
An experimental system to study cell cycle specific gene expression in plant cells was developed using protoplasts from tobacco cells synchronized by aphidicolin treatment. Chimeric plasmids consisting either of the chloramphenicol acetyltransferase (CAT) gene downstream of the cauliflower mosaic virus (CaMV) 35 S promoter or the nopaline synthase (nos) promoter were introduced into synchronized protoplasts of four cell cycle stages by electroporation. In the case of the CaMV 35 S promoter cyclic oscillation of CAT activity was observed which paralleled the cell cycle of the recipient cells. The peak of CAT activity was found in the S phase, while no such cyclic change was observed in the case of the nos promoter. This system clearly shows that it is feasible to search for a cell cycle specific promoter. The significance of these observations is discussed in relation to the study of plant cells.
The overlap of gametophytic and sporophytic gene expression in barley was studied by means of enzyme electrophoresis. Of the isozymes found, 60% were expressed in both gametophyte and sporophyte, 30% were sporophyte specific, and 10% were gametophyte specific. A considerable amount of the barley genome is thus potentially amenable to gametophytic selection. The estimated sizes of the common, sporophytic and gametophytic domains in barley gene expression correspond with the estimates obtained in other plant species.
Simultaneous coupling methods for detection of acid phosphatase and non-specific esterase produce a coloured reaction product that is quantitatively related to enzyme content in freeze-sectioned Brassica pollen and tapetal cells.
The intine-located acid phosphatase has 2 periods of synthesis: the first in late vacuolate period, associated with the completion of deposition of the intine polysaccharides; the second during pollen maturation, apparently reflecting cytoplasmic synthesis. Esterase activity accumulates in the tapetal cells until dissolution at early maturation period, when there is a dramatic rise in pollen-wall esterase activity, reflecting the transfer from tapetum to exine cavities. These quantitative studies confirm the gametophytic and sporophytic origins of the intine and exine proteins.
Plants represent a diverse group of organisms that have unique reproductive, developmental, and physiological processes. Although
morphologically simple, plants have molecular genetic processes that are equivalent in complexity to those found in animals.
Sophisticated gene transfer procedures, transposon mutagenesis in homologous and heterologous plants, and development of model
organisms such as Arabidopsis permit almost any gene that is associated with an observable phenotype to be isolated and studied.
These advances, coupled with general advances in molecular biology, now make it possible to dissect the molecular and cellular
events responsible for controlling plant-specific processes.
We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
An improved indigogenic method for the detection of β-glucuronidase was devised. The recommended medium consists of 2.4×10-4 M 4-Cl-5Br-3-indolyl-β-D-glucuronide (dissolved in a small amount of dimethylformamide) in 0,1 M acetate buffer pH 5, 3.4×10-3 M potassium ferricyanide and 3.4×10-3 M potassium ferrocyanide. The activity of β-glucuronidase escapes from unfixed or acetone postfixed cold microtome sections into the incubation solution and this leakage cannot be entirely prevented by either the postfixation of sections in cold aldehyde fixatives, or in acetone-formaldehyde-chloralhydrate mixture, or by fixation of freeze-dried sections in isopropylalcohol. No leakage was ascertained from sections prepared from tissue blocks fixed for 24 hours in cold aldehydes. This procedure leads to 70-80% inactivation of enzyme activity in rat spleen, liver and kidney, however. The recommended concentration of ferri-ferrocyanide mixture inhibits the activity by 30% at an average. The overall staining intensity obtained with the improved method in various organs reflects well activities of β-glucuronidase as estimated biochemically in homogenates of these organs. The method enables to demonstrate the lysosomal localization. The distribution pattern obtained with it resembles that obtained with the simultaneous azo-coupling method using naphthol AS-BI-β-glucuronide and hexazonium-p-rosanilin.
To isolate resistant cell lines, small pieces of callus (each weighing 50 or 250 mg) were planted on a selective medium containing a borderline streptomycin concentration (0.5 mg ml-1) according to the experiments of Binding4. After 6-8 weeks development of green shoots and proliferation of green callus could be observed in a few cases. Green tissues regularly appeared at a well defined region of inocula.
Summary During the meiotic prophase in pollen mother cells ofLilium species the ribosome population of the cytoplasm diminishes to a minimum reached during the diplotene-diakinesis interval. This fall is associated with a drop in the ribonuclease-extractable RNA present in whole, fixed cells. A rise in RNA and in number of observable ribosomes follows during the meiotic mitoses and in the very early life of the spores. Bodies with the characteristics of nucleoli—here termed “nucleoloids”—are released into the cytoplasm at anaphase I and anaphase II, and it is suggested that these may represent the main mechanism through which the ribosome population of the cytoplasm is restored after the prophase elimination.
One hundred and twelve Agrobacterium tumefaciens mutants with a deleted octopine Ti plasmid were isolated. They originated from four insertion mutants, each of which carried the transposon Tn904 at a different position in the Ti plasmid. The deletion mutants were selected on the basis of loss of the capacity encoded by the Ti plasmid to degrade octopine. They were tested for the expression of other Ti-plasmid coded functions: tumor induction, presence of lysopine dehydrogenase activity in the tumor, and exclusion of phage Ap-1. For 21 mutants affected in at least one of these functions, the map position of the deletions was determined. It was found that deletions at two separated loci give rise to an Occ− phenotype. Genes for Ap-1 exclusion were mapped on a small region just outside and to the right of the TL + TR region. Most of the TR region, present as TR-DNA in a limited number of crown gall tissues only, was shown to be unnecessary for tumor formation, since it could be deleted without affecting virulence (tested on various plant species) of the mutants. However, if the TR region together with a small part of the adjacent TL region, which is always present as TL-DNA in normal crown gall tissues, was deleted the mutants became weakly virulent on Kalanchoë and Nicotiana rustica and avirulent on tomato. We hypothesize that in this case a region necessary for T-DNA integration has been deleted. The same region was found to be essential for lysopine dehydrogenase activity in the tumors.
The available cytochemical methods for localization of beta-galactosidase have been evaluated using pollen grains of Brassica campestris. beta-Galactosidase-deficient pollen (gal), served as a control. Azo dye methods involving naphthyl substrates showed high and nonspecific background staining to the exine. The indigogenic method, employing 5-bromo-4-chloro-3-indoxyl beta-D-galactoside (X-gal) as the enzyme substrate, gave specific opaque-blue final reaction product, while mutant pollen grains remained colourless. Final reaction product formation was blocked by D-galactono-1,4-lactone, thus demonstrating the specificity of the enzyme reaction. Using microspectrophotometry, the absorbance of the final reaction product was found to be a linear function of incubation time and section thickness in cryostat sections up to 8 micron thick and was only slightly reduced by glutaraldehyde prefixation. The validity of the indigogenic method for quantitative analysis was confirmed by using an enzyme-containing polyacrylamide gel model system and enzyme-coupled Sepharose 4B beads. Cellular sites of enzymic activity have been determined using plastic sections: final reaction product occurred in the intine wall layer and peripheral cytoplasm.
A broad host range cloning vehicle that can be mobilized at high frequency into Gram-negative bacteria has been constructed from the naturally occurring antibiotic resistance plasmid RK2. The vehicle is 20 kilobase pairs in size, encodes tetracycline resistance, and contains two single restriction enzyme sites suitable for cloning. Mobilization is effected by a helper plasmid consisting of the RK2 transfer genes linked to a ColE1 replicon. By use of this plasmid vehicle, a gene bank of the DNA from a wild-type strain of Rhizobium meliloti has been constructed and established in Escherichia coli. One of the hybrid plasmids in the bank contains a DNA insert of approximately 26 kilobase pairs which has homology to the nitrogenase structural gene region of Klebsiella pneumoniae.
Arabinogalactan-proteins (AGPs), isolated from the pistils of Nicotiana alata, an ornamental tobacco, are developmentally regulated. Both the total amount and concentration of AGP in the stigma increase during flower development, reaching 10 micrograms AGP/stigma at maturity. In contrast, AGP concentration in the style remains constant throughout the maturation period reaching 12 micrograms AGP/style at maturity. The classes of AGP present in the stigma and style during flower development, separated according to their charge by crossed-electrophoresis, are different and change during development. Pollination of flowers of N. alata with compatible or incompatible pollen results in a significant and reproducible increase in the amount of AGPs in the stigma, but not the style, compared with control unpollinated pistils. Pollination with ethanol vapor inactivated pollen also results in an increase in the amount of AGP in the stigma, but this is less than half that observed following pollination with viable pollen. There are no significant differences in the classes of AGP, based on crossed-electrophoresis, present in the pistil following pollination.
Male gametophyte (pollen) isozyme profiles were compared with those of the sporophyte for nine enzyme systems. Sixty percent of the structural genes coding for these enzymes in the sporophyte were also found to be expressed by the gametophyte. All the genes tested were found to be expressed after meiosis, apparently transcribed and translated in the haploid gametophytes.
Overlap of gameto-phytic and sporophytic gene expression in barley The extent of gametophytic-sporophyfic gene expression in maize
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Pedersen S, Simonsen V, Loeschke V (1987) Overlap of gameto-phytic and sporophytic gene expression in barley. Theor Appl Genet 75 : 200-206 Sari Gorla M, Frova C, Binelli G, Ottaviano E (1986) The extent of gametophytic-sporophyfic gene expression in maize. Theor Appl Genet 72: 42-47
Quantitative cytoehemistry offl-galac-tosidase in normal and enzyme deficient (gal) pollen of Brassica
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Singh MB, Knox RB (1984) Quantitative cytoehemistry offl-galac-tosidase in normal and enzyme deficient (gal) pollen of Brassica 7: 225-228
Arabinogalactan-proteins of the female sexual tissue of Nicotiana alata
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Gell AC, Bacic A, Clarke AE (1986) Arabinogalactan-proteins of the female sexual tissue of Nicotiana alata. Plant Physiol 82:885-889
Pollen-pistil interactions. In: Linskens HF, Hes-lop-Harrison J (eds) Cellular interactions
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Knox RB (1984) Pollen-pistil interactions. In: Linskens HF, Hes-lop-Harrison J (eds) Cellular interactions. Encycl Plant Physiol 17: 508-608
Transmitting tissue and pollen tube growth. In: Linskens HF (ed) Fertilization in higher plants The Netherlands
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Indigogenic methods for esterases In: Danielli IF (ed) General cytochemical methods
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Indigogenic methods for esterases
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Arabinogalactan-proteins of the female sexual tissue of Nicotiana alata
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