Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl Transferase Gene.
TL;DR: The effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation, since no alterations in the programmed differentiation pattern were found with increased cytokinin levels.
Abstract: Cytokinins, a class of phytohormones, appear to play an important role in the processes of plant development. We genetically engineered the Agrobacterium tumefaciens isopentenyl transferase gene, placing it under control of a heat-inducible promoter (maize hsp70). The chimeric hsp70 isopentenyl transferase gene was transferred to tobacco and Arabidopsis plants. Heat induction of transgenic plants caused the isopentenyl transferase mRNA to accumulate and increased the level of zeatin 52-fold, zeatin riboside 23-fold, and zeatin riboside 5[prime]-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5[prime]-monophosphate in transgenic plants accumulated to levels 3 and 7 times, respectively, over levels in wild-type plants. This uninduced cytokinin increase affected various aspects of development. In tobacco, these effects included release of axillary buds, reduced stem and leaf area, and an underdeveloped root system. In Arabidopsis, reduction of root growth was also found. However, neither tobacco nor Arabidopsis transgenic plants showed any differences relative to wild-type plants in time of flowering. Unexpectedly, heat induction of cytokinins in transgenic plants produced no changes beyond those seen in the uninduced state. The lack of effect from heat-induced increases could be a result of the transient increases in cytokinin levels, direct or indirect induction of negating factor(s), or lack of a corresponding level of competent cellular factors. Overall, the effects of the increased levels of endogenous cytokinins in non-heat-shocked transgenic plants seemed to be confined to aspects of growth rather than differentiation. Since no alterations in the programmed differentiation pattern were found with increased cytokinin levels, this process may be controlled by components other than absolute cytokinin levels.
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TL;DR: This work has shown that in some late-flowering mutants of Arabidopsis, vernalization and an increase in the proportion of far-red light in the light source can substitute for one another in promoting the transition to flowering.
Abstract: The timing of the transition from vegetative growth to flowering is of paramount importance in agriculture, horticulture, and plant breeding because flowering is the first step of sexual reproduction. Studies to understand how this transition is controlled have occupied countless physiologists during the past half century and have produced an almost unmanageably large amount of information (Bernier et al., 1981a; Halevy, 1985-1989; Bernier, 1988; Kinet, 1993). A majority of plants use environmental cues to regulate the transition to flowering because all individuals of a species must flower synchronously for successful outcrossing and because all species must complete their sexual reproduction under favorable externa1 conditions. Any environmental variables exhibiting regular seasonal changes are potential factors that control the transition to flowering. The major factors are photoperiod, temperature, and water availability. Plants that do not require a particular photoperiod or temperature to flower, i.e., the so-called “autonomous-flowering” plants, are usually sensitive to irradiance. The environmental factors are perceived by different parts of the plant. Photoperiod and irradiance are perceived mainly by mature leaves in intact plants. Temperature is perceived by all plant parts, although low temperature (vernalization) is often perceived mainly by the shoot apex. Water availability is perceived by the root system. There are strong interactions between these different factors, so that each factor can change the threshold value for the effectiveness of the others. Plants, as opportunists, will thus make use of a different critical factor in different environments. Melilotus officinalis, for example, is a biennial with a vernalization requirement in temperate zones and an annual long-day (LD) plant with no cold requirement in arctic regions. In photoperiodic species, such as the short-day (SD) plant Pharbitis nil and the LD plant Silene armeria, flowering in unfavorable photoperiods can be caused by changing temperature, irradiance, or nutrition or by removing the roots. Similarly, in some late-flowering mutants of Arabidopsis, vernalization and an increase in the proportion of far-red light in the light source can substitute for one another in promoting the transition to flowering (Martinez-Zapater and Somerville, 1990; Bagnall, 1992). Clearly, there are alternate pathways to flowering in most, if
678 citations
TL;DR: The morphological phenotypes of some arr mutants suggest complex regulatory interactions and gene-specific functions among family members, suggesting a general involvement of type-A ARRs in light signal transduction.
Abstract: Type-A Arabidopsis (Arabidopsis thaliana) response regulators (ARRs) are a family of 10 genes that are rapidly induced by cytokinin and are highly similar to bacterial two-component response regulators. We have isolated T-DNA insertions in six of the type-A ARRs and constructed multiple insertional mutants, including the arr3,4,5,6,8,9 hextuple mutant. Single arr mutants were indistinguishable from the wild type in various cytokinin assays; double and higher order arr mutants showed progressively increasing sensitivity to cytokinin, indicating functional overlap among type-A ARRs and that these genes act as negative regulators of cytokinin responses. The induction of cytokinin primary response genes was amplified in arr mutants, indicating that the primary response to cytokinin is affected. Spatial patterns of ARR gene expression were consistent with partially redundant function of these genes in cytokinin signaling. The arr mutants show altered red light sensitivity, suggesting a general involvement of type-A ARRs in light signal transduction. Further, morphological phenotypes of some arr mutants suggest complex regulatory interactions and gene-specific functions among family members.
665 citations
Cites background from "Alterations of Endogenous Cytokinin..."
...Attempts to increase cytokinin levels by constitutive overexpression of bacterial isopentenyl transferases in whole plants resulted in no striking morphological effects because the plant may compensate for elevated biosynthesis by increasing the conjugation and degradation of the hormone (Medford et al., 1989; Smigocki, 1991; Mok and Mok, 2001a)....
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...Ectopic expression and overexpression of cytokinin biosynthetic genes have also demonstrated that elevated levels of cytokinin can release apical dominance, reduce root development, delay senescence, and enhance shoot regeneration in cultured tissues (Medford et al., 1989; Smigocki, 1991; Li et al., 1992; Gan and Amasino, 1995; Sa et al., 2001; Zubko et al., 2002)....
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...…have also demonstrated that elevated levels of cytokinin can release apical dominance, reduce root development, delay senescence, and enhance shoot regeneration in cultured tissues (Medford et al., 1989; Smigocki, 1991; Li et al., 1992; Gan and Amasino, 1995; Sa et al., 2001; Zubko et al., 2002)....
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...…has been linked to fundamental processes in plant growth and development, including the regulation of cell division, and altering endogenous cytokinin levels can have dramatic consequences on plant development and morphology (Miller et al., 1955, 1956; Medford et al., 1989; Werner et al., 2001)....
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...Cytokinin has been linked to fundamental processes in plant growth and development, including the regulation of cell division, and altering endogenous cytokinin levels can have dramatic consequences on plant development and morphology (Miller et al., 1955, 1956; Medford et al., 1989; Werner et al., 2001)....
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TL;DR: It was concluded that a major effect of PRD is the production of chemical signals in drying roots that are transported to the leaves where they bring about a reduction in stomatal conductance.
Abstract: Partial rootzone drying (PRD) is a new irrigation technique which improves the water use efficiency (by up to 50%) of wine grape production without significant crop reduction. The technique was developed on the basis of knowledge of the mechanisms controlling transpiration and requires that approximately half of the root system is always maintained in a dry or drying state while the remainder of the root system is irrigated. The wetted and dried sides of the root system are alternated on a 10-14 d cycle. Abscisic acid (ABA) concentration in the drying roots increases 10-fold, but ABA concentration in leaves of grapevines under PRD only increased by 60% compared with a fully irrigated control. Stomatal conductance of vines under PRD irrigation was significantly reduced when compared with vines receiving water to the entire root system. Grapevines from which water was withheld from the entire root system, on the other hand, show a similar reduction in stomatal conductance, but leaf ABA increased 5-fold compared with the fully irrigated control. PRD results in increased xylem sap ABA concentration and increased xylem sap pH, both of which are likely to result in a reduction in stomatal conductance. In addition, there was a reduction in zeatin and zeatin-riboside concentrations in roots, shoot tips and buds of 60, 50 and 70%, respectively, and this may contribute to the reduction in shoot growth and intensified apical dominance of vines under PRD irrigation. There is a nocturnal net flux of water from wetter roots to the roots in dry soil and this may assist in the distribution of chemical signals necessary to sustain the PRD effect. It was concluded that a major effect of PRD is the production of chemical signals in drying roots that are transported to the leaves where they bring about a reduction in stomatal conductance.
590 citations
TL;DR: The expression of ARR5 in the apical meristems was confirmed by whole mount in situ analysis of seedlings and is consistent with a role for cytokinin in regulating cell division in vivo, and is due, at least in part, to increased transcription.
Abstract: We examined the expression of a family of Arabidopsis response regulators (ARR) and found that the steady-state levels of RNA for most are elevated very rapidly by cytokinin. Using nuclear run-on assays we demonstrated that this increase in ARR transcript levels in response to cytokinin is due, at least in part, to increased transcription. The start site of transcription for the ARR5 gene was identified using primer extension analysis. A DNA fragment comprised of 1.6 kb upstream of the ARR5 transcript start site conferred cytokinin-inducible gene expression when fused to a beta-glucuronidase reporter, confirming that the transcription rate of ARR5 is elevated by cytokinin. This reporter construct was also used to examine the spatial pattern of ARR5 expression. The highest levels of expression were observed in the root and shoot apical meristems, at the junction of the pedicle and the silique, and in the central portion of mature roots. The expression of ARR5 in the apical meristems was confirmed by whole mount in situ analysis of seedlings and is consistent with a role for cytokinin in regulating cell division in vivo.
559 citations
01 Jan 1989
TL;DR: Control by Day Length, Genetics of Sensitivity to Environmental Factors, and Control by Day length.
Abstract: ENVIRONMENTAL CONTROL . . . . . . . . . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetics of Sensitivity to Environmental Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Control by Day Length . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
553 citations
References
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TL;DR: In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provide a basis for understanding how compartment-specific redox dynamics may operate in retrograde signaling and stress 67 acclimation in plants.
Abstract: In experiments with tobacco tissue cultured on White's modified medium (basal meditmi hi Tnhles 1 and 2) supplemenk'd with kiticthi and hidoleacctic acid, a slrikin^' fourlo (ive-told intTease iu yield was ohtaitu-d within a three to Tour week j^rowth period on addition of an aqtteotis exlrarl of tobacco leaves (Fi^'ures 1 and 2). Subse(iueutly it was found Ihiit this jnoniotiou oi' f^rowih was due mainly though nol entirely to inorj^auic rather than organic con.stitttenls in the extract. In the isolation of Rrowth factors from plant tissues and other sources inorj '̂anic salts are fre(|uently carried along with fhe organic fraclioits. When tissue cultures are used for bioassays, therefore, il is necessary lo lake into account increases in growth which may result from nutrient elements or other known constituents of the medium which may he present in the te.st materials. To minimize interference trom rontaminaitis of this type, an altempt has heen made to de\\eh)p a nieditmi with such adequate supplies of all re(iuired tnineral nutrients and cotntnott orgattic cottslitueitls that no apprecial»le change in growth rate or yield will result from the inlroduclion of additional amounts in the range ordinarily expected to be present in tnaterials to be assayed. As a point of referetice for this work some of the culture media in mc)st common current use will he cotisidered briefly. For ease of comparis4)n Iheir mineral compositions are listed in Tables 1 and 2. White's nutrient .solution, designed originally for excised root cultures, was based on Uspeuski and Uspetiskaia's medium for algae and Trelease and Trelease's micronutrieni solution. This medium also was employed successfully in the original cttltivation of callus from the tobacco Iiybrid Nicotiana gtauca x A', tanijadorffii, atitl as further modified by White in 194̂ ^ and by others it has been used for the
63,098 citations
"Alterations of Endogenous Cytokinin..." refers methods in this paper
...For measurements of root growth, sterilized seeds were germinated on agar medium (Murashige and Skoog, 1962) containing 30 g/L sucrose and Murashige and Skoog (MS) salts mixture (GIBCO) at one-half the normal concentration....
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TL;DR: GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage, and Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
Abstract: 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.
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TL;DR: The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine, which is applied to mutations introduced via both oligonucleotides and error-prone polymerization.
Abstract: Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.
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TL;DR: 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.
Abstract: 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 Agrobacter...
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TL;DR: All the advantages that nitrocellulose blotting previously afforded for DNA analysis are now available for RNA analysis as well.
Abstract: Publisher Summary The technique of E.M. Southern for transferring electrophoretically separated DNA fragments to nitrocellulose paper for subsequent hybridization with radioactive RNA or DNA probes has proved to be a powerful tool for the analysis of DNA. Until recently, the only comparable procedure for RNA has been to transfer and covalently couple it to activated cellulose paper (diazobenzyloxymethyl paper, DBM paper) according to the method of Alwine et al . Although DBM paper does offer the advantage of covalent attachment, it has several drawbacks as discussed in this chapter. The chapter also describes a related method for dotting RNA onto nitrocellulose pretreated with high salt. Thus, all the advantages that nitrocellulose blotting previously afforded for DNA analysis are now available for RNA analysis as well.
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