The Plant Cell, Vol. 1, 403-41 3, April 1989 O 1989 American Society of Plant Physiologists

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric lsopentenyl Transferase Gene

June 1. Medford,” Roger Horgan,b Zaki El-Sawi,b and Harry J. Klee”,’ a Plant Molecular Biology Group, Monsanto Company, 700 Chesterfield Village Parkway, St. Louis, Missouri 631 98

The University College of Wales, Department of Botany and Microbiology, Aberystwyth SY23 3DA, United Kingdom

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 hsp7O 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’-monophosphate twofold. At the control temperature zeatin riboside and zeatin riboside 5‘-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.

INTRODUCTION

In development, gene expression is coordinated in a tem- poral and spatial manner. Development in plants can be described by two processes. One process, growth, in- volves change in cell size and number. A second process, differentiation, encompasses modifications ranging from subtle changes in cellular metabolism to physiological and morphological modifications. The phytohormones appear to play an important role in regulating plant growth and differentiation. Evidence for this comes both from correla- tive studies, which attempt to relate endogenous hormone concentrations to specific physiological processes, and from effects of externally applied hormones. However, both lines of evidence are open to criticism. Correlative studies are frequently unable to distinguish between cause and effect, and the results of externa1 applications of hormones can only be interpreted correctly in relation to a number of incompletely understood phenomena. These

’ To whom correspondence should be addressed.

phenomena include mechanisms of uptake, transport, in- ter- and intracellular localization, the ability or inability of cells and tissues to respond to the phytohormone, and metabolic pathways leading to activation/deactivation. An approach to eliminating some of these problems is to develop a technology for directing in vivo production of phytohormones. This paper describes such an approach for cytokinins.

Cytokinins are a class of phytohormones first defined as substances that stimulate cell division in cultured tobacco cells. However, externally applied cytokinins exhibit a wide variety of effects. These include shoot initiation from callus cultures, promotion of axillary bud growth, directed trans- port of nutrients, stimulation of pigment synthesis, inhibi- tion of root growth, and delay of senescence. Given the problems with exogenous application and pleiotropic ef- fects, the possible role of endogenous cytokinins in plant growth and differentiation is difficult to assess.

Compounding the previously mentioned problems is a lack of knowledge concerning the regulation of cytokinin biosynthesis. In terms of sites of biosynthesis, high cyto-

404 The Plant Cell

kinin levels have been reported to occur in roots, immature leaves, and apical buds. However, demonstrating that these tissues are actual biosynthetic sites is difficult since the precise pathway of cytokinin production in plants has

RESULTS

Construction of a Heat-lnducible ipt Gene

not been rigorously established. In contrast, the pathway for cytokinin biosynthesis directed by the plant pathogen, Agrobacterium tumefaciens, is well established, The A.

A 700-bp fragment of the Inaize hsp70 prometer, including the entire 120-bp heat shock (hs) nontranslated leader, was fused to the ipt gene (Figure 1A). The nontranslated leader was included studies have sug- gested that there may be selected translation of heat shock genes during heat shock (McGarry and Lindquist, 1985). The chimeric gene was inserted into the binary plant transformation vectors pMON505 and pMON850 to create pMON595 and pMON601, respectively (Figure 1 B). Trans- genic tobacco and Arabidopsis plants were produced and DNA gel blot hybridization confirmed that the regenerated plants contained the hs-ipt gene (data not shown). Progeny

tumefaciens cytokinin biosynthetic gene codes for the enzyme isopentenyl transferase (ipt) (Akiyoshi et al., 1984; Barry et al., 1984). This enzyme catalyzes the condensa- tion of isopentenyl pyrophosphate and adenosine mono- phosphate giving isopentenyl AMP (iPMP) (McGaw, 1987). In tumorous plant tissue this enzyme may represent the rate-limiting step in cytokinin biosynthesis (Akiyoshi et al., 1983). Furthermore, this enzyme may have a role in non- tumorous tissues since a partially purified enzyme with ipt activity has been reported in tobacco cells (Chen and Melitz, 1979).

Although details of cytokinin biosynthesis are not clear, details relating to cytokinin metabolism and its role in regulating endogenous levels are somewhat clearer. There is some evidence that the free base may be the most biologically active form of cytokinin (Laloue and Pethe, 1982). Thus, most cytokinin metabolic processes lead to a reduction in biological activity (McGaw et al., 1984). However, the reversibility of some base/nucleoside/nu- cleotide modifications suggests that these may be endog- enous mechanisms for regulating the leve1 of the biolog- ically active cytokinins and thereby serve as a potential way to fine tune any type of developmental regulation. Some metabolic processes, such as side chain cleavage and N-glycosylation, are irreversible and result in removal of the cytokinins from the pool of biologically active mate- rial.

With the accessibility of cloned phytohormone biosyn- thetic genes from A. tumefaciens, endogenous cytokinin levels can be manipulated. Constitutive production of cy- tokinins from fusion of the ipt gene to a constitutive pro- moter produced shooty plants that failed to root (Klee et al., 1987). A system for regulated cytokinin production was designed to study better their role in plant development. One type of regulated expression is that of the heat shock genes. In response to elevated temperatures, organisms terminate transcription and translation of most proteins and initiate synthesis of a set of proteins known as the heat shock (hsp) proteins (Bond and Schlesinger, 1987; Key et al., 1987). Previously, Rochester et al. (1986) de- scribed the isolation of a maize hsp70 promoter, which allowed thermal induction of a chimeric gene in transgenic petunias. This promoter was fused to the A. tumefaciens ipt gene and the chimeric gene (hs-ipt) introduced into plants. We describe our experiments using thermal induc- tion of the hs-ipt gene to examine which processes of plant development are, and which processes are not, affected by alterations of endogenous cytokinin levels.

A. Transcription

BamHl Stari Bgl'l q , L d , - n - n - n 4 tis lsopentenyl Transferase I nontranslated

leader

B. Balll

pMON601 15766 pMON595 RB

13469

RB

u

Figure 1. Construction of the Chimeric hs-ipt Gene.

(A) Structure of the chimeric gene. A 700-bp fragment containing the maize hsp70 promoter and 5'-nontranslated leader sequence was fused to the ipt coding region from pTiT37. This promoter fragment contains two heat shock elements (HSE) located at -106 and -54 bp from the transcription start site. The non- translated leader consists of 120 bp derived from maize (\\\\\) and 20 bp of linker 3' to the Bglll site. (B) Plant transformation vectors, pMON595 and pMON601. Both vectors contain ColEl-type (pBR) and broad host range (RK2) origins allowing replication in Escherichia coli and A. tumefaciens, respectively. Each plasmid contains a 2.5-kb fragment of pTiT37 containing the T-DNA right border (RB) for integration into the plant genome and a marker gene, nopaline synthase (NOS). The spectinomycin resistance gene from Tn7 (Tn7 spc/str) allows selection in bacteria. Plasmid pMON595 contains a neomycin phosphotransferase gene (NOS/NPT/NOS) allowing kanamycin selection in plants, whereas pMON601 contains a gentamicin acetyltransferase AAC(3)-IV gene allowing gentamicin selection in plants.

Alteration of Endogenous Cytokinins 405

of the transgenic plants were identified by germinationassays on media with antibiotics (kanamycin for tobaccoor gentamicin for Arabidopsis) or by nopaline synthaseassays (Otten and Schilperoort, 1978). A. B.

Heat Induction of ipt Gene Expression

Based on data from preliminary experiments (Medford,Winter, and Klee, 1988), temperatures of 42°C (Arabidop-sis) and 45°C (tobacco) were chosen. Arabidopsis andtobacco plants were heat shocked in vivo for 2 hr, andimmediately afterward, the hs-ipt mRNA could be readilydetected (Figure 2). In the uninduced transgenic tobaccoplants, the ipt message could not be detected, even with20 jug of poly A+ RNA per lane and extensive exposure ofautoradiograms (Figure 2B). This indicates that the levelsof uninduced ipt mRNA in transgenic plants must be lessthan 1.0% of the induced levels.

Heat Induction of Cytokinins in Transgenic Plants

The transgenic plants were examined for heat-inducedaccumulation of Cytokinins. Plants were heat shocked andallowed to recover for 4 hr, and tissue was harvested,frozen in liquid nitrogen, and lyophilized. The major cyto-kinins previously identified in tobacco crown gall tissue(containing the ipt gene) were measured using the 2H/15Ntechnique of Scott and Morgan (1984). iPMP and thecytokinin nucleotide, ZMP (ribosyl zeatin 5'-monophos-phate) were measured by selective ion monitoring using2H internal standards whereas Z (zeatin, the free base),ZR (ribosyl zeatin), and Z7G (zeatin 7-0-o-glucoside) weremeasured using 15N internal standards. No precautionswere taken to minimize phosphatase action during extrac-tions since the method used can account for this. However,recovery of the 2H-iPMP internal standard was generallyvery low, suggesting that the values for the level of thiscompound are probably underestimates.

The level of all Cytokinins measured was considerablyelevated in heat-shocked transgenic plants (Table 1). Thelevel of Z increased 52-fold, ZR increased 23-fold, andZMP increased twofold. In addition, the presence of aconsiderable quantity of Z7G in the heat-shocked plantsindicates that a significant amount of metabolic inactivationof cytokinins occurred in the 4 hr following heat shock.Relative to cytokinin levels found in tobacco crown galltissues, the base (Z) and riboside (ZR) are much higher inthe transgenic heat-shocked plants. The most likely reasonfor this is that the production of cytokinins in the transgenicplants takes the form of a rapid biosynthesis pulse inducedby the heat shock, whereas in crown gall tissues, cytoki-nins are produced by a slower continuous process.

870 bp 870 bp

Figure 2. Analysis of ipt mRNA Expression in Transgenic Plants.

Transgenic plants were heat-shocked at 42°C (Arabidopsis) or45°C (tobacco) for 2 hr. RNA was isolated immediately after heatshock.(A) 40 M9 of total RNA isolated from transgenic Arabidopsis plantsat the control and heat-shocked temperature. The arrow indicatesthe 870-bp chimeric ipt mRNA.(B) Twenty micrograms of poly A+ RNA isolated from transgenictobacco plants at the control and heat-shocked temperature.

It is of interest to note that, in transgenic plants main-tained at the control temperature, the levels of ZR andZMP are elevated (threefold and sevenfold, respectively).This is indicative of a slight "leakiness" of the gene at thecontrol temperature. Although this was not observable atthe mRNA level, a small increase in cytokinin productionintegrated over a period of time could result in the in-creased levels of ZR and ZMP (Table 1).

Effects of Cytokinin Alterations on Growth andDifferentiation

Changes in the Shoot System of Tobacco

Transgenic tobacco plants were examined for effects dueto altered cytokinin levels. Even without thermal induction,tobacco plants containing the hs-ipt gene showed a reduc-tion in height (Table 2). However, the number of nodes inwild-type and transgenic plants was not significantly dif-ferent. Thus, whether the height reduction is a result ofdecreased number of nodes, internode length, or a com-bination thereof, could not be clearly determined. In thetransgenic plants there was a reduction in xylem content.Mature xylem in transgenic plants represented only 70%

406 The Plant Cell

Table 1. Cytokinin Levels in Wild-Type and Transgenic Tobacco Plants

Cytokinin Level, ng/g

iPMP Z ZR ZMP Z7G

Wild-type 14 46 44 86 ND" Wild-type, heat-shocked 18 18 48 72 ND Transgenic 31 23 94 640 ND Transgenic, heat-shocked 102 1450 2230 1667 700 ND

Plants were heat shocked for 2 hr. Four hours afterwards, cytokinins were extracted from leaf tissues and quantitated as described in Methods. a ND, not determined.

of the controls. In a similar pattern, leaf size in the trans- genic plants was reduced, representing only 73% of the controls.

Heat inductions were given at weekly intervals, starting when the plants had four true leaves greater than 3 cm in length. At this point the plants have formed only 1 O to 12 of their 30 to 40 nodes. Even in the absence of heat induction, transgenic plants again showed a reduction in height (Figure 3). In response to heat induction, wild-type plants showed no changes in height or other morphological factors. Unexpectedly, heat induction of cytokinins in the transgenic plants produced no changes beyond those seen in the uninduced state (Figure 3). These results, with effects in uninduced plants and no further effects in in- duced plants, were observed in several independent trans- genic plants.

Cytokinins have been thought to enhance the onset of flowering in several species (Bernier, 1988). In the trans- genic tobacco plants, despite the increased leve1 of cyto- kinins from uninduced or induced production, there was no significant change in the days to flowering (Table 2). In addition to flowering, the enhanced production of cytoki- nins in uninduced transgenic plants did not perturb devel- opment of fertilized ovules into seeds. In heat-shocked plants, both wild-type and transgenic, the elevated tem- perature itself (45OC) caused ovule abortion.

Two other aspects of cytokinin alterations on the pattern of plant growth were noted. First, axillary bud growth in the transgenic plants was significantly greater than that of wild-type plants (Figure 4, A and B). Although the greatest differences were found at the uppermost nodes, this dif- ference was true for all nodes of the plant (Figure 4C). A second aspect of plant growth, heterostylic development (which was previously reported in grafted shoots contain- ing the ipt gene [Wullems et al., 1981]), was notably absent in the uninduced transgenic plants. After heat induction, heterostylic development was found, but since this was true for both wild-type and transgenic plants, it was im- possible to distinguish between the effects from heat shock and effects from elevated cytokinins.

Changes in the Shoot System in Arabidopsis

Heat induction of cytokinins in Arabidopsis started with plants 7 days after sowing (16-hr photoperiod) and at 3- day intervals thereafter, up to the point of anthesis. As in tobacco, the thermal induction of cytokinins in transgenic Arabidopsis plants produced no further changes in the development of the shoot system. Thermal induction of cytokinins was performed throughout floral development. No differences in floral differentiation that could be attrib- uted to cytokinin overproduction alone were observed. As in tobacco, there was no effect of uninduced cytokinins on ovule development, and heat induction at 42OC produced abortion in both the transgenic and control plants.

Arabidopsis, a facultative long day plant, grown under long days (1 6 hr) will go from seed to flowering in 6 weeks. Transgenic plants grown under these conditions showed no discernible reduction in xylem proliferation as seen in transgenic tobacco plants. However, under short day (8 hr) conditions, Arabidopsis plants required 5.5 months to go from seedling to flowering stage. When floral stem cross sections of short day plants were examined, the transgenic Arabidopsis plants had reduced xylem prolifer- ation similar to that found in the transgenic tobacco plants (Figure 5).

Table 2. Comparison of Wild-Type and Transgenic Tobacco Plants

Transgenic Wild-Type

Node number 35.1 -C 1.7 36.9 f 2.2 Stem area 3.0 C 0.7 4.1 f 0.8 Days to flowering 71.9 f 2.6 72.6 f 4.9

Measurements were done on F, progeny from a cross of a transgenic plant to a wild-type plant. Transgenic progeny were identified by nopaline synthase assays. Details of measurements are found in Methods.

Height, cm 80.3 f 5.9 94.7 f 12.0

Alteration of Endogenous Cytokinins 407

80

h

60 v

CI r w Q, I o

*- 40 c a ul v) c

I-

.-

E 20

O O 2 4 6

Number of Heat Shocks Figure 3. Reduction of Height in Tobacco Plants Containing the hs-ipt Construct.

Heights of individual plants were measured at the first anthesis. Bars indicate standard deviation. Each point consists of data from at least six plants.

In Arabidopsis, exogenous application of cytokinins has been reported to enhance the onset of flowering (Mich- niewicz and Kamienska, 1965; Besnard-Wilbaut, 1981). To test the effect of enhanced cytokinin levels on flowering, plants were grown in short (8-hr) days. Two and a half months after sowing, one set (wild-type and transgenic) was given heat shocks every 3rd day for a period of 3 weeks. Heat shocks were conducted at times so as not to disrupt the photoperiod. Two months after the conclu- sion of the heat induction (5.5 months from the start of the experiment), the plants flowered. However, there was no significant difference in the time of flowering between plants containing the hs-ipt gene and wild-type plants. This was true for both those plants exposed to thermal induc- tion and those maintained at the control temperature.

An additional effect of cytokinin overproduction in non- heat-shocked tobacco plants was the enhanced develop- ment of axillary buds. Axillary growth in transgenic Arabi- dopsis plants grown in long days was somewhat enhanced in the rosette nodes, although the extremely small size

made conclusions difficult. Plants grown in short days were not examined for axillary bud growth.

Changes in the Root System of Transgenic Plants

In transgenic tobacco and Arabidopsis plants, the primary root elongated at a slower rate than that of wild-type plants (70% for tobacco, 68% for Arabidopsis). In both species lateral roots were initiated in a pattern indistin- guishable between transgenic and wild-type plants. How- ever, the root tip region of transgenic plants (Figure 6, A and B) was wider and shorter than that of wild-type plants. In addition, root hairs in transgenic plants emerged closer to the tip. This suggests that the zone of elongation is reduced in transgenic plants. In wild-type plants, longitu- dinal files of maturing rectangular cells could be easily traced. However, in the transgenic plants, the periclinal walls of the maturing cells were often extended, causing a disruption of the orderly pattern of growth (Figure 6A). This pattern of growth was not readily discernible in the young seedling roots, but became more obvious as the roots aged. At maturity, this effect resulted in a reduction of root mass (Figure 7).

DISCUSSION

Cytokinins are believed to play regulatory roles in two processes that describe plant development: growth and differentiation (Finkelstein et al., 1987; King, 1988). Eluci- dation of processes that cytokinins affect has come, in part, from studies where the hormone is applied externally. This approach presents added complexities that limit de- finitive conclusions. This is problematic since it is equally important to understand the processes that are not ef- fected by phytohormones as well as those that are eff ected.

To regulate endogenous cytokinin levels, we fused an ipt gene from A. tumefaciens to a maize hsp7O promoter. After heat induction, the ipt mRNA was readily detectable in transformed Arabidopsis and tobacco plants (Figure 2). Although the mRNA could not be detected without heat induction, the threefold and sevenfold increase in the levels of ZR and ZMP (respectively) in the uninduced transgenic plants indicates that the chimeric gene must be expressed to some extent at control temperatures. Although expres- sion of the maize hsp70 promoter at control temperatures must be nominal, even a low leve1 of constitutive ipt gene expression at control temperatures could account for the accumulation of cytokinins, since expression of ipt mRNA from the promoter normally found in the A. tumefaciens Ti plasmid (a relatively weak promoter) is enough to alter plant morphology (Akiyoshi et al., 1983).

408 The Plant Cell

9 12 15 18 21

NODE NUMBER (from apex)

Figure 4. Comparison of Growth of Axillary Buds in Wild-Type and Transgenic Tobacco Plants.(A) and (B) Growth of axillary bud at 12th node (from shoot apex) in wild-type (top) and transgenic (bottom) plants.(C) Fresh weight (mg) of axillary buds from nodes of wild-type and transgenic plants.

Presumably, the uninduced expression from the maizehsp70 gene occurs equally throughout the plant. To inves-tigate tissue distribution of the heat shock response, thehsp70 promoter was fused to the reporter gene, 0-glucu-ronidase (GUS). At the control temperature, no detectableGUS activity was found in transgenic tobacco and Arabi-dopsis plants containing the hs-GUS fusion. However, 4hr after heat induction, histochemical analysis showedGUS expression in all major organs and tissues (data notshown).

Accumulation of cytokinins (Table 1) demonstrates thatthe chimeric hs-ipt gene is producing a functional enzyme,and that the plant is converting iPMP into the cytokinins,Z, ZR, and ZMP. Despite large increases in cytokinin levelsfollowing heat induction, no further effects beyond thoseseen at the control temperature were observed. This wasunexpected since our similar experiments with the samepromoter and auxin biosynthetic genes showed dramaticalterations in phenotype after heat induction (Medford andKlee, 1988). There are several possible explanations, none

Alteration of Endogenous Cytokinins 409

Figure 5. Cross-section of Wild-Type and Transgenic ArabidopsisFloral Stems Grown in Short Days.

Wild-type stems (top) and transgenic stems (bottom) stained withtoluidine blue (magnification x50).

of which are mutually exclusive. First, since the increasedcytokinin level is most likely transient, it is possible thatthe altered levels of active cytokinins do not persist longenough for a phenotypic response to be observed. Asecond possibility is that there may be a direct or indirectinduction of negating factors such as metabolic enzymeslike cytokinin oxidase. The high level of Z7G 4 hr after heatshock supports an enhancement of cytokinin metabolism.A third possibility is that there is a lack of correspondinglevel of cellular factors for perception of the high level ofcytokinins.

In uninduced transgenic plants, the elevated cytokininlevels altered some aspects of plant development. In thetransgenic plants, there was a reduction in height, leafarea, and stem width. The reduced stem width correlated

with xylem reduction in both transgenic tobacco plantsand transgenic Arabidopsis plants grown in short days. InArabidopsis plants grown in long days, this effect was lesspronounced. Since Arabidopsis plants mature rapidly un-der long day conditions, a small reduction in xylem prolif-eration would not be discernible. However, growth ofArabidopsis plants under short days takes 5 to 6 monthsand would allow small changes in xylem formation tobecome more apparent.

In addition to changes in the amount of xylem, axillarybud growth in the transgenic tobacco and Arabidopsisplants was increased. This agrees with exogenous appli-cation studies where cytokinins were applied directly tothe axillary buds (Tamas, 1987). Our previous studies onauxin overexpression in transgenic petunias (Klee et al.,1987; Medford and Klee, 1988) suggested that auxinsuppression of axillary buds is a result of auxin levelsrather than gradients. Our work here indicates that in-creased cytokinin levels enhanced the growth of the axil-

Figure 6. Effect of Increased Cytokinin Levels on Root Growth.

(A) Arabidopsis primary root at 14 days. Right, wild-type; left,transgenic.(B) Tobacco primary root at 12 days. Left, wild-type; right, trans-genic (magnification of both figures is x50).

410 The Plant Cell

Figure 7. Cumulative Effects of Cytokinin Alterations on TobaccoRoot Growth at Maturity.

Left, wild-type; right, transgenic.

lary buds. Collectively, these data indicate that axillary budrelease is dependent on the auxin/cytokinin ratio withinthe bud.

In both tobacco and Arabidopsis, the increase in cytokin-ins (both uninduced and induced) had no effect on flower-ing. We are unaware of any direct report of the effect ofcytokinins on flowering in tobacco. However, in Arabidop-sis, two independent studies have shown that applicationof exogenous cytokinin (kinetin) to the shoot apex resultedin enhanced floral development (Michniewicz and Kamien-ska, 1965; Besnard-Wilbaut, 1981). The work of Besnard-Wilbaut (1981) showed that, for kinetin to be effective atfloral induction, the plants first had to reach a certaindevelopmental stage. In our studies, the elevated level ofcytokinins under non-heat-shocked conditions is presum-ably constant throughout development. In addition, heat-induced elevations of cytokinins were done when theplants should have been competent to respond to thecytokinin increase. The major difference between our studyand previous ones is that the endogenous productionpresumably occurs throughout the entire plant, whereasthe exogenous cytokinins were applied directly to theshoot apex. Thus, enhancement of flowering in Arabidop-sis by exogenous cytokinins could be more a result ofselective nutrient redistribution to a competent apex ratherthan a specific action of cytokinin per se. Alternatively, itis possible that, in the transgenic Arabidopsis plants, therewas only a partial evocation, which would require extensiveanalysis of the shoot tip to distinguish.

The effect of altered cytokinins on the root system wassimilar in both tobacco and Arabidopsis. Root growth,measured in terms of elongation, was reduced in thetransgenic plants. The reduction in the rate of root growthcorresponded to a decrease in the size of the zone of

elongation. A previous study has shown a correlationbetween reduced size of the zone of elongation and re-duced root growth rate (Hinchee, 1981). Another effect ofaltered cytokinin levels on root growth in transgenic plantswas a disruption of the orderly pattern of longitudinal filesof cells. The similar phenomenon was observed in vitrowhen kinetin was supplied exogenously to maize rootsand was attributed to dissolution of the middle lamella ofcell walls parallel to the root surface (Kappler and Kristen,1986). In the transgenic plants, we noted that these wallswere often bulging, consistent with the exogenous appli-cation study. In the transgenic plants this effect becamemore conspicuous as the roots grew. Since disruption ofcell organization was observed only in the roots, thissuggests that the root system may be more sensitive toalterations in cytokinin levels than the shoot system.

Cytokinins have been described as compounds that, incombination with auxin, delineate a differentiation pathway(Wareing and Phillips, 1981). We noted that the majordevelopmental effects of altered cytokinin levels in thetransgenic plants were on aspects of plant growth and notdifferentiation. Since these changes were found in a num-ber of tissues (e.g., leaves, stems, roots), the mechanismby which cytokinins bring about growth modifications maybe a fundamental change in aspects of cellular metabolism.In altering the endogenous cytokinin levels in the trans-genic plants, no alterations in the differentiation processeswere detected. We could not find any evidence for changesin the plant's programmed pattern of differentiation. Thiswas true in both the shoot system and the root systemfrom two different plant families. The fact that the alteredendogenous cytokinin levels per se did not affect differ-entiation suggests that this process, unlike the process ofplant growth, may be controlled by components other thanabsolute cytokinin levels.

METHODS

Vector Construction

A 700-bp fragment from the maize hsp70 promoter (Rochester etal., 1986) was cloned into pUC119 for site-directed mutagenesisusing the procedure described by Kunkel (1985). A Bglll site wasintroduced at the translational start site. This allowed the entirepromoter and 5'-nontranslated leader to be excised as a BamHI-Bglll fragment. This fragment was then cloned into the uniqueBglll site of pMON505 (Horsch and Klee, 1986), creatingpMON591. The ipt gene from pTiT37 (Goldberg et al., 1984) wascloned into pMON591 as a 1.2-kb Bglll-EcoRI fragment, creatingpMON595 (Figure 1). The same fragments were inserted into theplant transformation vector pMON850, creating the plasmidpMON601 (Figure 1). For construction of the /i-glucuronidasegene (GUS) fusion, the mutagenized heat shock promoter wasfused to a 1.9-kb Hindlll fragment from pMON9749 (Hinchee etal., 1988) containing the entire GUS opening reading frame fusedto the nopaline synthase 3' sequence.

Alteration of Endogenous Cytokinins 41 1

Plant Materials Heat-shocked tissue was placed in the substrate solution 4 hr after return to the control temperature.

Nicotiana tabacum cv Samsun and Arabidopsis thaliana ecotypes Columbia and RLD were grown in controlled environmental cham- bers at 25OC, 50% relative humidity, and 16-hr day length. To- bacco plants received water and nutrients twice a day from automatic watering systems, whereas Arabidopsis plants were watered by hand and fertilized once a week. For one set of experiments with tobacco, plants were grown in the winter months in a greenhouse maintained at 25'C with supplemental lighting. Greenhouse-grown plants were watered daily by hand and re- ceived a fertilizer once a week. For experiments on flowering in Arabidopsis, plants were grown in a Percival set for short days (8 hr) with watering and fertilization by hand when required.

Thermal lnductions

Heat shocks were performed in vivo by temperature increases in a controlled environmental chamber. To use the heat shock promoter to create inducible changes in endogenous phytohor- mone levels, conditions were first defined that allowed both gene induction in vivo and plant survival. Plants were heat-shocked by a linear elevation of temperature over a 15-min period. The elevated temperature was maintained for 2 hr with relative humid- ity of 70%, followed by a 15-min linear decline. The plants (both Arabidopsis and tobacco) survived the heat shock at all temper- atures tested.

Plant Transformation RNA Gel Blot Analysis

Tobacco plants transformed with pMON595 were obtained using the leaf disc method (Horsch et al., 1985). Arabidopsis plants transformed with pMON601 were obtained using the method of Lloyd et al. (1986) with modifications for gentamicin selection (Hayford et al., 1988). Progeny of primary transformants were identified by germinating seed on 1 O0 pg/mL kanamycin (tobacco) or 80 pg/mL gentamicin (Arabidopsis).

Growth Measurements

For measurements of root growth, sterilized seeds were germi- nated 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. Petri dishes were placed vertically and the length of the primary root measured after 5 days for Arabidopsis. For tobacco, the primary root was marked 5 days after germination and the distance from the initial mark to the root tip measured at 12 days. Growth at the root tip was observed directly or after brief staining in toluidine blue.

Tobacco leaf areas was measured as a product of the lamina length and its width at the widest part (6 cm from the base). Stem area was determined by tracing the stem cross-sections at node 14, followed by weight measurements of the tracing. Stems were sectioned by hand, stained with toluidine blue, and examined under a microscope. Measurements of xylem proliferation were done at nodes 7 and 14 on 12 separate plants. The extent of xylem proliferation was determined by counting the mature ele- ments in an individual file of cells. Height and cross-sectional area determinations were done on a population of transgenic and wild- type plants. Stem cross-sectional area was calculated by tracing the stem diameter at the 15th node (from the base) on paper followed by weighing the paper. Results were analyzed using the Student's t test and were significant to P > 0.995.

GUS Histochemical Assays

Histochemical assays were performed using X-gluc (5-bromo-4- chloro-3-indolyl-p-~-glucuronic acid, cyclohexylammonium salt, Research Organics) as a substrate (Jefferson et al., 1987). Tissues were sectioned by hand, placed into the substrate solution, vac- uum infiltrated, and incubated at room temperature overnight.

RNA was isolated from plant tissue using precipitation with LiCl (Rochester et al., 1986). Poly A+ RNA was prepared using oligo(dT) column chromatography as described (Maniatis et al., 1982). RNA was separated on 1.4% agarose gels with formalde- hyde and transferred to a nylon membrane (Thomas, 1983). Expression of ipt mRNA was analyzed by hybridization of the cloned ipt gene to the RNA gel blots. Hybridizations were done at 42°C for 16 hr in 50% formamide, 4 x SSPE, 4 x Denhardt's solution, 1% SDS, and tRNA at 100 pg/mL (Maniatis et al., 1982). The filters were washed twice for 20 min at room temperature in 0.5 x SSPE, 0.1% SDS followed by one wash at 65OC in the same solution.

Cytokinin Analysis

Plants were heat-shocked as described above and 4 hr afterward, leaf tissue (tobacco) or the entire rosette (Arabidopsis) was ex- cised, frozen immediately in liquid nitrogen, and lyophilized. The lyophilized tissues were extracted in 80% methanol with the addition of 'H2-ZMP and iPMP, and '5N4-Z, ZR, and Z7G as the interna1 standards. The cytokinins were purified by reverse-phase HPLC and measured by GC-MS-selective ion monitoring as the permethyl derivatives, with the exception of Z7G, which was analyzed as the trimethylsilyl derivative. The method used was essentially that of Scott and Horgan (1 984).

ACKNOWLEDGMENTS

We thank Jill Winter and Maud Hinchee for their helpful discus- sions. We thank Nancy Hoffmann for producing the transformed tobacco plants and Yvonne Muskopf for assistance with trans- genic Arabidopsis plants. We are also grateful to Steve Rogers, Rob Horsch, and Robb Fraley who provide continuous support to all our efforts.

Received February 6, 1989.

41 2 The Plant Cell

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DOI 10.1105/tpc.1.4.403 1989;1;403-413Plant Cell

J. I. Medford, R. Horgan, Z. El-Sawi and H. J. KleeTransferase Gene.

Alterations of Endogenous Cytokinins in Transgenic Plants Using a Chimeric Isopentenyl

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