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First published online July 25, 2002; 10.1105/tpc.003186 American Society of Plant Biologists PtABI3 Impinges on the Growth and Differentiation of Embryonic Leaves during Bud Set in Poplar
a Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium 1 To whom correspondence should be addressed. E-mail woboe{at}gengenp.rug.ac.be; fax 32-(0)9-264-5349
The Arabidopsis ABSCISIC ACID–INSENSITIVE3 (ABI3) protein plays a crucial role during late seed development and has an additional function at the vegetative meristem, particularly during periods of growth-arresting conditions and quiescence. Here, we show that the ABI3 homolog of poplar (PtABI3) is expressed in buds during natural bud set. Expression occurs clearly after perception of the critical daylength that initiates bud set and dormancy in poplar. In short-day conditions mimicking natural bud set, the expression of a chimeric PtABI3::  -glucuronidase (GUS) gene occurred in those organs and cells of the apex that grow actively but will undergo arrest: the young embryonic leaves, the subapical meristem, and the procambial strands. If PtABI3 is overexpressed or downregulated, bud development in short-day conditions is altered. Constitutive overexpression of PtABI3 resulted in apical buds with large embryonic leaves and small stipules, whereas in antisense lines, bud scales were large and leaves were small. Thus, PtABI3 influences the size and ratio of embryonic leaves and bud scales/stipules that differentiate from the primordia under short-day conditions. These observations, together with the expression of PtABI3::GUS in embryonic leaves but not in bud scales/stipules, support the idea that wild-type PtABI3 is required for the relative growth rate and differentiation of embryonic leaves inside the bud. These experiments reveal that ABI3 plays a role in the cellular differentiation of vegetative tissues, in addition to its function in seeds.
Plants continuously adjust the growth rate of their organs in response to developmental and environmental signals. One possible means of integrating the growth of different organs consists of imposing dormancy on some of the meristems along the plant. This strategy is elaborated most evidently by trees that arrest many of their meristems in axillary buds to achieve their typical architecture. Bud dormancy is exploited similarly when plants have to persist through periods that are unfavorable for growth or that are potentially hazardous, such as the winter season. In this case, growth inactivity is imposed on all meristems.
Despite many physiological studies, only fragmentary knowledge exists regarding the endogenous processes that direct the creation of a dormant state (Lang, 1996
One ideal candidate to test this hypothesis is the ABSCISIC ACID–INSENSITIVE3 (ABI3) gene, which plays a crucial role during late seed development (Bonetta and McCourt, 1998
Despite the facts that the most prominent phenotype of the abi3 mutant is its reduced seed dormancy and the prevalent function of ABI3 is in seeds (Parcy et al., 1994
Here, we have investigated the function of the ABI3 homolog from poplar (Populus trichocarpa), PtABI3, throughout bud development and dormancy. Although poplars do not develop a very deep dormancy in buds and seeds, they have been used successfully to study the genetics and physiology of bud dormancy (Bradshaw and Stettler, 1995 In short days, Populus tremula x Populus alba trees transformed with sense and antisense constructs of PtABI3 showed altered bud morphology. Constitutive overexpression of PtABI3 resulted in apical buds with large embryonic leaves and small stipules, whereas in antisense lines, bud scales were large and leaves were small. Thus, PtABI3 influences the size and ratio of embryonic leaves and bud scales/stipules that differentiate from the primordia under short-day conditions. These observations, together with the expression of PtABI3::GUS in embryonic leaves but not in bud scales/stipules, suggest that wild-type PtABI3 is required for the relative growth rate and differentiation of embryonic leaves during bud set. ABA levels peaked in buds concomitantly with PtABI3::GUS expression, suggesting that ABA and PtABI3 act simultaneously in bud set. We conclude that PtABI3 is an essential component of successful bud set that in turn is a precondition for the establishment of dormancy.
PtABI3 Is Expressed during Autumnal Bud Set under Natural Conditions To delineate the seasonal expression pattern of PtABI3, its expression was analyzed in various tissues and in apical buds. A bud, surrounded by protective bud scales, contains a densely packed series of young leaves and leaf primordia that do not elongate but undergo growth arrest (approximately from August until March). By contrast, an active apex produces leaves that elongate simultaneously (approximately from April until August). PtABI3 expression, as shown in Figure 1 , was observed in developing poplar seeds, consistent with the prominent expression of ABI3 in Arabidopsis seeds. In apical buds, PtABI3 was found to be expressed exclusively in September, which corresponds to the time of vegetative bud set under natural conditions (Figure 1). Because bud set covers a 2-month period in field conditions (Belgium, 50°N) from the perception of the critical daylength (15.5 h) by mid August until the formation of the terminal bud by mid October, these data suggest that PtABI3 is not involved directly in the perception of the critical daylength. PtABI3 expression has not been analyzed from April until July because no apical buds are made during these months. However, PtABI3::GUS analyses never revealed expression in actively growing apices, which are equivalent to summer month apices (see below).
Interestingly, the expression of PtABI3 in autumn buds coincided with that of PtABI1b, as it did in seeds (Figure 1). ABI1 is a negative regulator of ABA signal transduction in Arabidopsis seeds. The simultaneous expression of PtABI3 and PtABI1b in buds at the time of growth arrest shows that seed and bud dormancy involve, at least in part, similar molecular components. However, unlike PtABI3, PtABI1b was expressed in buds at other times and in leaves and xylem of actively growing plants. Weak PtABI3 expression also was found in the xylem of greenhouse-grown P. tremula x P. alba (Figure 1). Because the expression in buds of PtABI3, but not of PtABI1b, appeared exclusively during autumnal bud set, we characterized its expression with a chimeric promoter-GUS fusion (PtABI3::GUS) and studied its function during bud set in transgenic P. tremula x P. alba upregulated and downregulated for PtABI3 expression.
Bud Set under Controlled Short-Day Conditions
During the first 3 weeks under short days, all primordia that had been committed before the onset of short days will develop into leaves, although internode elongation between them ceases gradually and becomes fully arrested after 3 to 4 weeks (Figure 3A, primordia 5 to 0). The last primordium that had been committed before the onset of short days is the last leaf to mature, often not to its full size, and to subtend the bud (Figure 3A, organ 0). The stipules of this leaf may persist after leaf abscission and enlarge to serve as bud scales for the developing bud. Only the primordia initiated after the perception of the critical daylength will change morphogenetically from the beginning of their development (Figure 3A, primordium -1 and younger ones). After the 4th week, the bud scales are apparent (Figure 2B). They originate from the first primordium initiated after the onset of short days: the lamina of this primordium senesces and abscises prematurely, so that the stipular domains of the primordium enlarge to form bud scales (Figure 3A, abortive bud scale leaf). Embryonic leaves immediately distal to the abortive bud scale leaf do not abort their lamina. Nevertheless, the development of their midrib is suppressed so that the lamina appears broad and short (Figure 3A, incipient bud scale leaf). Inside the forming bud, organogenesis proceeds to form embryonic leaves sensu stricto, each with two stipules, and leaf primordia without yet distinct stipules (Figure 3A, embryonic leaves sensu stricto and leaf primordia).
Although all organs inside a bud often are commonly called leaf primordia, Goffinet and Larson (1981)
For convenience, we refer to bud scales, stipules, and embryonic leaves as separate organs, although they may have arisen from a single primordium. Moreover, the term "embryonic leaves" is used in a broader sense than that suggested by Goffinet and Larson (1981) After these organs have been made, the activity of the apical meristem ceases and the bud is formed (Figure 2C). Besides the described morphogenetic processes, some aspects of natural bud set are not accomplished under short-day treatments. P. tremula x P. alba plants, for example, do not shed leaves and do not acclimate to cold under the conditions used (see Methods). Bud set is a precondition for the establishment of dormancy in the meristem; still, considerable time may elapse before full winter dormancy is achieved.
PtABI3::GUS Expression in Buds under Short-Day and Long-Day Conditions
Soon after the visible appearance of bud scales, PtABI3::GUS expression was detected in apical buds. As shown in the micrographs in Figure 4
, the highest expression of PtABI3::GUS was seen in the youngest embryonic leaves inside the setting apical bud, starting at
Furthermore, PtABI3::GUS activity was observed in the procambial strands and in the cambium of the first two internodes below the apical bud (Figures 4A, 4F, and 4G). The expression in these tissues coincided with young, actively growing cells whose growth has to be retarded and arrested to ultimately meet short-day conditions. Additionally, expression was detected in single cells or small groups of cells close to the vascular cambium (Figure 4H). These cells often contained a large oxalate crystal, indicating their marked differentiation compared with neighboring cells. This aspect of PtABI3::GUS expression is interesting because it might highlight a link with cell-autonomous differences in the state of cellular differentiation. PtABI3::GUS expression lasted from the 4th week after the onset of short days until the end of the short-day experiment (6 weeks). When plants were returned to long days after various periods of short-day treatment, PtABI3::GUS expression persisted in the inactive buds, presumably because of GUS protein stability, but disappeared as soon as growth had resumed (data not shown). Axillary buds on long-day plants also change during their development from an active phase of organ initiation to an inactive, dormant state. In accordance with the fact that these buds also go through a developmental phase that prepares their arrest and dormancy in long-day plants, PtABI3::GUS activity also was detected in the axillary buds (data not shown). Similarly, cambial PtABI3::GUS expression also occurred in actively growing long-day plants (data not shown). In both cases, the expression pattern was completely comparable to that of axillary buds and cambium in the short-day plants shown in Figure 4. However, in axillary buds and cambium of long-day plants, the expression was found at a greater distance from the apex than in plants grown under short days. In both cases, moreover, this distance varied greatly among individual plants, probably depending on vigor and environmental conditions. None of these stages of PtABI3::GUS expression under long days could be correlated with obvious phenotypic alterations in PtABI3-downregulated or PtABI3-overexpressing P. tremula x P. alba, suggesting that PtABI3 acts along with additional factors that are present only in growth-arresting short days. In conclusion, the cessation of internode elongation and bud scale initiation happened well before the time when PtABI3::GUS expression was detected in the embryonic leaves. Therefore, PtABI3 functions during embryonic leaf growth and differentiation but not during leaf initiation. Moreover, the expression occurred in organs and cells in which growth still occurs but ultimately needs to be stopped: the young embryonic leaves, the subapical meristem, and the procambial strands.
PtABI3 Overexpression Prevents Correct Bud Formation in Response to Short Days
The growth response of overexpressing and control plants was comparable during the first 4 weeks after transfer to short-day conditions. On average, four additional internodes, which accounted for an Figure 5 gives a detailed analysis of the correspondingly aberrant bud morphology in terms of embryonic leaves versus stipules/bud scales. After 6 weeks in short days, wild-type buds formed typical bud scales that encased entirely the embryonic leaves and their stipules (Figures 5A and 5E). By contrast, overexpression of PtABI3 led to plants with larger embryonic leaves with clear vascular bundles and small stipules, leaving the arrested apex exposed in an open bud (Figures 5B and 5F). There were no bud scales encasing the bud (Figures 5B and 5F). This apex morphology of PtABI3-overexpressing plants resembled that of actively growing plants (cf. Figures 2D and 2F with 5D and 5F).
As shown in Figure 6 , not only embryonic leaf growth and differentiation had been favored in PtABI3-overexpressing plants, but the number of embryonic leaves also had increased slightly (Figure 6A). Compared with the wild type, the increased number of leaf organs underscores the significance of the morphological observation that embryonic leaf differentiation is favored in the PtABI3-overexpressing lines. In its target tissue, the young embryonic leaves in which wild-type PtABI3 normally is expressed, PtABI3 overexpression allowed prolonged growth and differentiation compared with the wild type. On the other hand, in nontarget tissues in which wild-type PtABI3 normally is not expressed, the growth of bud scales and stipules was suppressed to varying degrees in different overexpression lines (Figures 5F and 6A).
Another distinct characteristic of the PtABI3-overexpressing plants is that an enormous amount of starch accumulated in the buds, as seen in the electron micrographs shown in Figure 7 . Numerous amyloplasts were observed in the PtABI3-overexpressing plants, whereas wild-type plants had only small chloroplasts with a few membranes and occasionally a tiny starch granule (Figures 7A to 7D). This observation shows once more that PtABI3 overexpression alters the metabolic state of both the embryonic leaves and the stipules.
Concomitant with the changes in bud morphology, changes in the vascular bundle immediately beneath the bud were noted. The stem tissue of the first internode below the bud of PtABI3-overexpressing plants clearly was different from that of wild-type plants. Figure 8 illustrates the morphological differences found in comparable stem segments. As stated above, wild-type and PtABI3-overexpressing plants differed neither in height nor in time of elongation arrest. Although the wild type achieved vascular maturity up to the apical bud, the vascular bundle of overexpressing plants remained in a less developed state (Figures 8A and 8B).
In the wild type, the differentiation of xylem vessel cells and phloem fibers was found  2800 and 3200 µm below the apex, respectively (Figure 8C). Neither morphological characteristic was found at a distance of <4000 µm below the apex in the two overexpressing lines (n = 5 for both) that were investigated in detail (Figure 8D). Because wild-type PtABI3 was expressed in procambial strands (Figures 1, 4A, and 4F), the less mature vascular bundle of PtABI3-overexpressing plants has to be attributed to an altered, probably slower, differentiation of this tissue under overexpression conditions. However, the possibility cannot be excluded that this vascular phenotype is a consequence of altered leaf development in the open bud of the PtABI3-overexpressing lines or vice versa. The phenotypic changes in the overexpression lines correlated with a significant overexpression of the PtABI3 transcript in the apical buds, as illustrated for lines S8 and S16 in Figure 9
.
PtABI3 Antisense Lines Display Pronounced Bud Scale Development Because a pronounced growth and differentiation of embryonic leaves out of a primordium had been observed in PtABI3-overexpressing plants, PtABI3 antisense plants were expected to display opposite phenotypes. Although RT-PCR easily revealed the expression of the PtABI3 antisense transcript, it remained difficult to demonstrate the downregulation of the endogenous PtABI3 transcript, because it was impossible to design primers that specifically detected the endogenous transcript (Figure 9). Nevertheless, 4 of 16 tested independent antisense lines showed similar and reproducible phenotypic alterations that were not observed in the wild type. In these PtABI3 antisense lines, the growth of bud scales was very pronounced, occasionally even leading to a greater number of bud scales (Figures 5C, 5G, and 6A). The embryonic leaves tended to be smaller and fewer than those of the wild type (Figures 5C and 6A). However, the bud scales and embryonic leaves did not differ from those of the wild type in starch accumulation or in ultrastructure (data not shown). That the growth and differentiation of embryonic leaves cannot be finalized because of the absence of PtABI3 and are arrested prematurely may be the reason why the bud scales grow and differentiate more extensively than in the wild type. The advanced bud scale differentiation can be inferred from the accumulation of resinous compounds, as is apparent from the heavily staining inclusions in the outer bud scales of the antisense plants (Figure 5G). Furthermore, unlike what was seen in PtABI3-overexpressing poplars, the formation of xylem vessels and phloem fibers in the vascular bundle beneath the bud took place earlier than in the wild type. Xylem vessels and phloem fibers, although morphologically similar to those of the wild type (Figures 8A and 8C), were found closer to the apex in the two antisense lines (n = 5 for both) studied in detail (at 2000 and 2500 µm instead of 2800 and 3200 µm in the wild type). Thus, in the absence of PtABI3, vascular differentiation proceeds faster than in the wild type. If the alterations in organ number in PtABI3-overexpressing and antisense plants are expressed as an index of stipules per leaf, it becomes evident that PtABI3 influences the ratio of embryonic leaves and bud scales/stipules that differentiate out of the primordia under short-day conditions (Figure 6B). Comparing the bud morphology of PtABI3-overexpressing and antisense plants (Figures 5F and 5G) suggests the most obvious differences in bud scales. However, the expression of PtABI3::GUS occurs in embryonic leaves and not in bud scales/stipules, leading to the conclusion that wild-type PtABI3 is required primarily for the relative growth rate and differentiation of the embryonic leaves and, as a compensating effect, for the growth and differentiation of the stipules and bud scales.
ABA Concentration Shows a Distinct Peak during Short Day–Induced Bud Set
The timing of the increase in ABA concentration could not be correlated with changes in environmental factors, because photoperiod, temperature, humidity, and watering of the plants had been kept constant. For that reason, this ABA peak was inherent to the developmental program of bud set. Moreover, it coincided with the inception of bud scales and the onset of PtABI3::GUS expression. Therefore, PtABI3 may act with ABA in preparing bud set.
Bud and Seed Dormancy Share ABI1- and ABI3-Mediated Signal Transduction Pathways Seed and bud dormancy have common physiological characteristics that have led to the assumption that both are based on similar processes (Wareing, 1956  ). Extending the physiological analogies, both seed and bud dormancy also could involve similar cellular and molecular mechanisms. In support of this hypothesis, three of five identified PtABI1 genes and the PtABI3 gene, their Arabidopsis homologs being crucial regulators during late seed development (Leung and Giraudat, 1998), were found to be expressed both in seeds and during bud set in poplar (Figure 1; A. Rohde, unpublished data). The map position of one of the three PtABI1 genes expressed at bud set, PtABI1b (Figure 1), was found to coincide with a quantitative trait locus affecting bud set and bud flush in a poplar mapping pedigree designed to maximize the segregation of dormancy-related traits (Frewen et al., 2000). From these results, the idea emerges that at least ABI1 and ABI3 play a role during both bud set and seed dormancy.
Moreover, the PtABI3 expression data clearly suggest a function of ABI3 in meristem differentiation in both seed and vegetative tissues that are prepared for dormancy. Although seeds are regarded as the most prevalent dormant organ, the bud probably was the first dormant organ to evolve during evolution (Crabbé, 1994 Seed and bud dormancy both involve ABI3 expression, but the analogy with ABI3 function applies only at the molecular level, not at the level of the whole dormancy process. Obviously, ABI3 is integrated with different molecular networks in the two processes, implying that ABI3 may have different functions. It remains to be determined whether ABI3 expression in buds and seeds depends on the same or different developmental and environmental factors.
PtABI3 Promotes the Growth and Differentiation of Embryonic Leaves in the Bud before Growth Arrest Both of these morphogenetic events happen well before the time that PtABI3::GUS expression is first detected. PtABI3 is expressed exclusively in organs and cells that grow actively but will undergo growth arrest: the young embryonic leaves, the subapical meristem, and the procambial strands (Figure 4). Growth in these tissues has to be finalized and arrested and acclimation has to be initiated to prepare the plant for the expected environmental changes signaled by short days. As revealed in the embryonic leaves, PtABI3 impinges on the embryonic leaf development that has been initiated before. Most likely, in the other tissues in which PtABI3 is expressed, PtABI3 also functions during differentiation toward a particular fate, perhaps through slowing of growth cessation. That PtABI3 has a role in embryonic leaf growth and differentiation is supported by the phenotypes of PtABI3-overexpressing and antisense plants. Overexpression of PtABI3 resulted in plants with larger embryonic leaves and smaller bud scales than those in the wild type, leaving the apex exposed in an open but nevertheless arrested bud (Figures 2 and 5). The relative growth and differentiation rate of an embryonic leaf out of a primordium is favored over that of a bud scale or a stipule (Figure 6). By contrast, in antisense lines, bud scale growth and differentiation were more pronounced and the embryonic leaves inside the bud tended to be smaller than in the wild type (Figure 5). Thus, the downregulation of PtABI3 leads to the suppression of part of the leaf differentiation program and is compensated for by bud scale development. These observations suggest that wild-type PtABI3 is required for the relative growth rate and differentiation of the embryonic leaves and, correlated with it, of the bud scales and stipules, well before their eventual growth arrest. Therefore, the expression of PtABI3 controls the final differentiation state that is achieved in the embryonic leaves before their further development is arrested and interrupted by dormancy. At first glance, the fact that the downregulation of PtABI3 leads to an apparently dormant bud, whereas bud formation is not accomplished by PtABI3 overexpression (Figure 5), suggests that PtABI3 promotes leaf growth rather than dormancy. However, meristematic activity finally is arrested in both PtABI3 overexpression and antisense lines, but the degree of embryonic leaf growth and differentiation is different. Proper leaf differentiation is as much a part of successful bud set as is the development of encasing bud scales.
Is the Function of ABI3 Conserved in Poplar and Arabidopsis?
For example, although the expression of ABI3 and PtABI3 seems to be well conserved in seeds (Figure 1) (Parcy et al., 1994
Moreover, because of the duration of bud set, the expression of PtABI3 could be correlated with a particular phase during bud set, namely the preparation of embryonic leaf tissues for dormancy before bud set and dormancy. These functional aspects cannot be revealed during the establishment of vegetative quiescence in Arabidopsis. Because quiescence is short and difficult to control in Arabidopsis, ABI3 expression was correlated only with meristematic growth arrest in general (Rohde et al., 1999
At the functional level, PtABI3-overexpressing poplars seem to proceed with "growth" and thus to resemble the prematurely germinating abi3 mutant much more than PtABI3 antisense poplars do. However, a closer look reveals that the growth of the abi3 mutant is a consequence of skipped seed maturation as much as the "nongrowth" of PtABI3 antisense poplars results from the omission of part of embryonic leaf differentiation. Overexpression phenotypes remain conflicting. PtABI3 overexpression led to prolonged embryonic leaf differentiation, whereas ABI3 overexpression had no phenotypic consequences during seed germination (Parcy et al., 1994
ABI3 overexpression renders Arabidopsis plants hypersensitive to ABA but does not lead to constitutive ABA-related responses (Parcy et al., 1994 In conclusion, a functional analogy of PtABI3 and ABI3 exists at the molecular and cellular level, but it cannot be taken to the gross phenotypic level. Yet, like ABI3, PtABI3 also acts in the maturation and differentiation of cells and tissues or allows the time for it to occur. PtABI3 acts on appropriate growth and differentiation of embryonic leaves before bud set and dormancy, as ABI3 acts on the appropriate accumulation of storage proteins and the acquisition of desiccation tolerance during seed dormancy.
PtABI3 and ABA Act Simultaneously on Bud Set
One such candidate could be ABA. Indeed, ABA often has been found in dormant tissues and has growth-arresting effects when applied exogenously. ABA concentrations in apical buds increased significantly after
The fact that ABA alone cannot provoke ABI3/PtABI3 expression in Arabidopsis (Parcy et al., 1994
However, the rather close interaction between ABA and PtABI3 presumed in this scenario will remain hypothetical until the precise relationship of ABI3 and ABA is elucidated. Interesting in this respect is the fact that Arabidopsis ABI3 interacts with ABI5, an ABA-inducible TRAB homolog (transcription factor responsible for ABA regulation), and identifies a cross-point of ABI3-mediated and ABA-mediated signal transduction (Finkelstein and Lynch, 2000
Another important observation is the prominent starch accumulation found exclusively in PtABI3-overexpressing plants (Figure 7). Similarly, Arabidopsis overexpressing ABI3 plants were shown recently to be hypersensitive to Glc (Finkelstein and Gibson, 2001
PtABI3 Is an Essential Component of Bud Set
Plant Material and Growth Conditions Apical buds were harvested monthly (1995 and 1998) and additionally weekly from August 21 until October 14 (1998) from 3-year-old trees of poplar (Populus trichocarpa cv Trichobel) grown in the field (Institute of Forestry and Game Management, Geraardsbergen, Belgium). At each sampling time, the apical buds of all branches ( 20 buds) of three trees were harvested and pooled per tree. Poplar seeds were generated from a controlled cross in the greenhouse between clone 70,039/115 and clone 70,039/109 of Populus trichocarpa and harvested at the indicated days after pollination (Figure 1). The pollen of the male clone was collected and stored at 4°C until female flower buds were perceptive for pollination.
For all experiments with transgenic plants, Populus tremula x Populus alba clone INRA No. 717.1B4 was used. Selected transgenic lines were multiplied clonally in vitro on Murashige and Skoog (1962)
Gene Isolation and Reverse Transcriptase–Mediated PCR
For the amplification of ABI1 homologs from poplar, degenerated oligonucleotide primers for nested PCR amplification were designed based on the ABI1 sequence of Arabidopsis thaliana (Leung et al., 1994 Homologous primers were synthesized on the basis of the poplar sequence and led to the identification of the PtABI1b homolog. Its predicted protein is 64% similar to the ABI1 and ABI2 proteins. In total, five poplar ABI1 homologs have been identified to date, with PtABI1b being the most homologous with the ABI1 and ABI2 proteins. For each of these homologs, specific primers for PCR amplification have been developed. The following sense and antisense primers were used for PtABI1b: 5'-CGTATCCATTCGGCTTTGT-3' and 5'-GATCCACTGATAATGCCATT-3', resulting in a fragment of 303 bp on cDNA. For the amplification of the actin homolog, degenerated oligonucleotide primers were designed based on an alignment of several known actin genes from plants. The degenerated primers 5'-GGG-AYGAYATGGARAARACNTGG-3' and 5'-GGNGCNCANACYTTN-GTYTTCAT-3' were complementary in the sense and antisense directions to the sequences that encode the conserved amino acids WDDMEKTW and MKTKVVAP, respectively. A 582-bp fragment was amplified from genomic DNA of poplar and sequenced. On the basis of this sequence, homologous primers (5'-TGGCATCACACCTTCTAC-3' and 5'-CGACCACTGGCATAAAGG-3' for the sense and antisense directions, respectively) were used for the amplification of a 190-bp fragment.
Total RNA from various tissues of poplar was extracted according to Goormachtig et al. (1995) In the other RT-PCR experiments (Figure 9), reactions in 25 µL (50 ng of each primer) contained a modified nucleotide mix: dCTP, dTTP, and dGTP were at 200 pmol, whereas dATP was reduced to 20 pmol. To each reaction, 0.1 µL of 33P-labeled dATP (10 mCi/mL, 2500 Ci/mmol) was added, resulting in a hot-to-cold dATP ratio of 1:2500. Products were separated on 4.5% polyacrylamide gels and visualized on dried gels by means of autoradiography.
ABI3 Constructs and the Production of Transgenic Poplar
For the generation of PtABI3 sense and antisense constructs, the PtABI3 cDNA (Rohde et al., 1998
Constructs 35S::ABI3s, 35S::ABI3as, and PtABI3::GUS were transformed into Agrobacterium tumefaciens strain C58RifR(pMP90) by heat shock (Zahm et al., 1984 In these experiments, five overexpression lines (S8, S11, S16, S19, and S24) and four antisense lines (AS3, AS18, AS22, and AS29) were selected for further detailed study. Of the 22 PtABI3::GUS lines tested under short days, 16 expressed PtABI3::GUS. Seven of these lines with high and temporally and spatially consistent expression (4B, 11A, 14A, 15B, 16A, 17A, and 17B) were selected for the experiments. To correlate either the overexpression or the reduced expression of PtABI3 with the observed phenotypic changes, transcripts of the endogenous PtABI3 and the transgenes were followed by RT-PCR (Figure 9). 33P-dATP PCR conditions were as described above. The following primers were used as sense and antisense primers for the amplification of the endogenous, sense, and antisense transcripts, respectively: 5'-TCTTACCACAGGAATCTGAATCAT-3' and 5'-GGGCCAAAACCTATAACGCAT-3' (460 bp); 5'-TGCGTTATAGGT-TTTGGCCCA-3' and 5'-CGAGCTCTCCCATATGGTCGACCTG-3' (310 bp); and 5'-GCCCAAGCAGCCGCTGAAGAAG-3' and 5'-GTG-ATTTCAGCGTACCGAATTCC-3' (370 bp). For the sense and antisense transcripts, one of the primers was constructed so that the sequence was complementary to the junction of the PtABI3 cDNA, with the subsequent vector sequence in front of the CaMV 3' untranslated region.
Histochemical GUS Assays
Light Microscopy For GUS-stained material, selected vibroslices were treated as described above and embedded in Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany) after the 95% ethanol step according to the manufacturer's instructions. Sections (4 µm) were cut on a rotary microtome and mounted. Sections were examined with dark-field optics.
Transmission Electron Microscopy Ultrathin sections of gold interference color were cut using an ultramicrotome and collected on collodion-coated copper grids of 200 mesh. The sections were poststained in an ultrostainer (Leica, Herburgg, Switzerland) for 15 min in 2% uranyl acetate (Ultrostain 1; Leica) at 40°C and for 4 min in lead citrate (Ultrostain 2; Leica) at 20°C. The sections were examined by transmission electron microscopy (Elmiskop 101; Siemens, Karlsruhe, Germany).
Analysis of Endogenous Abscisic Acid and Indole-3-Acetic Acid
Abscisic acid (ABA) and indole-3-acetic acid (IAA) in the apical bud and the first leaf of individual plants were analyzed using a combined solid-phase extraction procedure (Prinsen et al., 1991
Accession Numbers Upon request, all novel material described in this article will be made available in a timely manner for noncommercial purposes. No restrictions or conditions will be placed on the use of any material described in this article that would limit their use for noncommercial research purposes.
The Institute of Forestry and Game Management (Geraardsbergen, Belgium) is gratefully acknowledged for all field material of poplar. We thank Bart Burggraeve and Sevgi Öden for technical assistance, Martine De Cock for help in preparing the manuscript, and Rebecca Verbanck and Karel Spruyt for artwork. A.R. is indebted to the Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie (Brussels, Belgium) for a postdoctoral fellowship.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.003186. Received March 19, 2002; accepted May 8, 2002.
Arenas-Huertero, F., Arroyo, A., Zhou, L., Sheen, J., and León, P. (2000). Analysis of Arabidopsis glucose insensitive mutants, gin5 and gin6, reveals a central role of the plant hormone ABA in the regulation of plant vegetative development by sugar. Genes Dev. 14, 2085–2096. |
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Abstract
INTRODUCTION
-bromopentafluorotoluene (Netting and Milborrow, 1988