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Proc Natl Acad Sci U S A. Jan 22, 2002; 99(2): 1064–1069.
PMCID: PMC117430
Plant Biology

The tomato Blind gene encodes a MYB transcription factor that controls the formation of lateral meristems

Abstract

The multitude of forms observed in flowering plants is largely because of their ability to establish new axes of growth during postembryonic development. This process is initiated by the formation of secondary meristems that develop into vegetative or reproductive branches. In the blind and torosa mutants of tomato, initiation of lateral meristems is blocked during shoot and inflorescence development, leading to a strong reduction in the number of lateral axes. In this study, it is shown that blind and torosa are allelic. The Blind gene has been isolated by positional cloning, and it was found that the mutant phenotype is caused by a loss of function of an R2R3 class Myb gene. RNA interference-induced blind phenocopies confirmed the identity of the isolated gene. Double mutant analysis shows that Blind acts in a novel pathway different from the one to which the previously identified Lateral suppressor gene belongs. The findings reported add a new class of transcription factors to the group of genes controlling lateral meristem initiation and reveal a previously uncharacterized function of R2R3 Myb genes.

In flowering plants, postembryonic shoot development is controlled by the activity of the shoot apical meristem (SAM). The SAM established during embryogenesis produces in a regular fashion leaf, node, and internode primordia, which generate the primary shoot of a plant. Secondary meristems arise in the axils of leaves as well as on the flanks of the inflorescence meristems. Lateral meristems produced during the vegetative phase develop into shoots repeating, at least in part, the growth pattern of the primary shoot, whereas after floral transition lateral meristems develop into flowers or new inflorescence axes.

In Arabidopsis thaliana, Zea mays, and several other plant species, genes have been isolated that are required for SAM initiation, maintenance, and function, and interactions between these genes are being studied (1). However, much less is known about the genetic control of lateral meristem initiation and function during shoot and inflorescence development. In tomato, lateral meristems are formed in all leaf axils and are first detectable in the axil of the fifth youngest primordium (2). They develop into fast-growing side shoots that give the plant a bushy appearance. Whereas in many higher plants the primary SAM remains active throughout the entire lifespan, in tomato, it is transformed into a terminal inflorescence, and the uppermost axillary meristem takes over its function to continue the main stem. After formation of three leaves, this sympodial shoot terminates itself in an inflorescence, and sympodial shoots of progressively higher order elongate the main axis (3, 4). The tomato inflorescence has been described as a cyme, i.e., flowers arise as terminal structures, and the inflorescence axis grows because of a lateral meristem, which will again be transformed into a floral meristem and so on (3). Recent analysis has in part modified this view by demonstrating that the inflorescence meristem is split into two halves, a floral and an infloral meristem, the last one reiterating the same developmental steps (5).

Mutants affected in the process of lateral shoot formation have been found in different plant species and can be divided into two groups. In mutants of the first group, like the Arabidopsis mutants auxin resistant 1 (6) and supershoot (7), as well as the maize teosinte branched mutant (8), growth of lateral meristems and the resulting buds is enhanced in comparison to the wild type. Mutants of the second group are characterized by a defective lateral meristem initiation. In the Arabidopsis revoluta (rev) mutant, formation of axillary meristems is inhibited in the majority of leaf axils. In rev mutants, initiation of floral meristems seems not to be impaired, but some of the flowers have a reduced number of organs because of a reduced floral meristem size (9, 10). In the tomato lateral suppressor (ls) mutant, formation of lateral meristems is almost completely blocked during vegetative development. The side shoots in the two leaf axils preceding an inflorescence are nevertheless usually formed, and branching of ls inflorescences is only slightly reduced (11). Additional tomato mutants of this group are blind (bl) and torosa (to), which are affected in the formation of all types of lateral meristems; during vegetative development, axillary meristems are not formed in many leaf axils (12), and also the sympodial shoot and the side shoot below are affected, although at a lower frequency. bl and to inflorescences are strongly reduced, consisting of only one or few flowers, which are often fused (1315).

Characterization of genes required for lateral meristem initiation will help to elucidate the control of shoot branching. As a first member of this group, the Ls gene has been isolated (16) and was shown to encode a protein of the GRAS family. This group of plant-specific putative transcription factors includes the Arabidopsis genes GIBBERRELIN INSENSITIVE (17) and REPRESSOR OF ga1–3 (18), two negative regulators of GA signaling, as well as SCARECROW (19) and SHORTROOT (20), two regulators of root development. The Arabidopsis REV gene was shown to belong to a subfamily of plant homeodomain/leucine zipper genes containing a putative sterol binding domain (21, 22).

In this study, data are provided showing that bl and to mutants are affected in the same gene. The Bl gene was cloned, and it was shown to belong to the R2R3 class of Myb genes, one of the largest plant transcription factor families (23), participating in quite different regulatory pathways (24).

Materials and Methods

Plant Materials.

Tomato seed material of Lycopersicon esculentum bl-1 (LA0059), bl-2 (LA0980), ls (LA0329), self pruning (sp, LA3133), and the wild-type cv. Moneymaker was obtained from the Tomato Genetics Resource Center (Davis, CA). Tomato seeds of L. esculentum cv. Condine Red, Condine Red-to-1, Lukullus, and Lukullus-to-2 were obtained from the Genebank, Institute for Plant Genetics and Crop Plant Research (Gatersleben, Germany). According to the stock list of the Tomato Genetics Resource Center, to-2 has been renamed bl-2. Plants were grown under standard glasshouse conditions with additional artificial light (16-h photoperiod) during the winter period.

DNA Isolation and Southern Blot Analysis.

Plant DNA preparation and Southern blot analysis have been described (25). Standard techniques were carried out according to Sambrook and Russell (26), unless otherwise stated.

Restriction Fragment Length Polymorphism (RFLP) Linkage Analysis.

RFLP linkage analysis was as described (16). The RFLP markers TG46, TG546, and TG36 (27) and several yeast artificial chromosome (YAC) end sequences were converted into cleaved amplified polymorphic sequence (CAPS) markers. F2 plants with recombination breakpoints within the TG46-TG36 interval were selected by a PCR-based approach; plants that are homozygous for the L. esculentum allele for one of the markers, but carry an Lycopersicon pennellii allele for one of the other markers, are informative with respect to the map position of blind.

RNA Isolation and Reverse Transcription (RT)-PCR Analysis.

Total RNA was isolated by using the RNeasy plant mini kit (Qiagen, Chatsworth, CA) following the manufacturer's instructions. For RT-PCR analysis, 1 μg of total RNA was reverse transcribed by using the Superscript II polymerase (Life Technologies, Grand Island, NY) according to the manufacturer's instructions. The product of the first-strand cDNA synthesis reaction was amplified by PCR by using the Bl-specific primers c79–40 (position 59–80 of cDNA) and c79–41 (position 1007–981). Using the primers 5′-ACTGGAATGGTTAAGGCTGGA-3′ and 5′-TCTGAACCTCTCTGCTCCAA-3′ specific for the potato gene PoAc101 (28), amplification of actin cDNA was performed as a control to ensure that similar amounts of cDNA were added to each PCR reaction. PCR products were detected by hybridization to radiolabeled probes.

Rapid Amplification of cDNA Ends (RACE).

RACE experiments were performed by using the RACE system of Life Technologies. For 5′ RACE, first-strand cDNA synthesis was performed with the specific primer c79–41. A first PCR was carried out with the universal adapter primer BRL-UAP and c79–35 (position 219–196), nested PCR with the abridged universal adapter primer BRL-AUAP and c79–20 (position 185–162). For 3′ RACE, first-strand cDNA was synthesized with the adapter-oligo-dT-primer BRL-AP. A first PCR was carried out with BRL-UAP and c79–43 (position 343–362), nested PCR with BRL-UAP and c79–24 (position 568–590). Amplified fragments were cloned into the pGEM-T (Promega) plasmid vector and sequenced.

Isolation and Characterization of YAC Clones.

Primers derived from the RFLP markers TG546 and TG46 as well as primers specific for the left end of YAC154 were used for screening a tomato YAC library (29, 30). Ends of the YAC inserts were amplified by inverted PCR (IPCR, ref. 31), subcloned in pGEM-T and sequenced. YAC end probes were either used directly for RFLP mapping or were converted into CAPS markers before mapping.

Establishment of a Cosmid and Plasmid Contig.

Cosmid clones were isolated from a genomic library prepared from L. esculentum cv. Moneymaker as described (16). A plasmid library was obtained by cloning double-digested DNA (KpnI/PstI, PstI/SstI, and KpnI/SstI) from the yeast strain harboring YAC313 in pUC19 and pGEM11Zf(+). Clones from this library were screened as described (16). The contig covering the segment between Cos30 and P10 (Fig. (Fig.2)2) was completely sequenced by using a primer walking strategy.

Figure 2
Map-based cloning of the Blind gene. (A) RFLP map and physical map of the region spanning the blind locus (not drawn to scale). YAC clones are shown as horizontal lines. Mapped YAC ends are indicated by the extension R or L. For mapped markers, the numbers ...

Tomato Transformation.

For inhibiting the function of the Bl gene by RNA interference (RNAi, ref. 32), fragments of the cDNA from positions 59–110, 804–373, and 250-1007 were assembled and introduced into the vector pRT-Ω/Not/Asc (33). The cassette containing the Bl sequences between the cauliflower mosaic virus 35S promoter and polyA site was inserted into the binary vector pGPTV-Kan-AscI (33) and transferred into the Agrobacterium tumefaciens strain GV3101 (34). Transformation of tomato leaf explants was done as described (35).

DNA Sequencing and Analysis.

DNA sequencing was done by using the PRISM Ready Reaction Terminator Cycle Sequencing system (Applied Biosystems). Reactions were run on an Applied Biosystems 373A or 377XL DNA sequencer. Computer analysis was performed by using the software WISCONSIN PACKAGE version 10.0, Genetics Computer Group (Madison, WI). Putative coding regions were identified by translated BLAST searches (blastx, tblastx; ref. 36) against NCBI sequence databases (nonredundant, expressed sequence tags).

Results

Phenotypic and Genetic Analysis of blind and torosa Mutants.

The tomato mutants blind-1 (bl-1), blind-2 (bl-2), and torosa-1 (to-1) are characterized by phenotypic abnormalities at different stages of development. During vegetative development, axillary buds are not formed in 40–90% of their leaf axils (Fig. (Fig.1),1), whereas wild-type plants develop side shoots in more than 99% of their axils. Decapitation experiments (37) and histological analysis (12) indicate that the lack of lateral shoots is because of a defect in lateral meristem initiation. bl and to plants develop lateral buds preferentially between the 2nd and the 5th node of the primary shoot and on the two nodes below the inflorescence (12). Some of these side shoots grow out much later than in wild-type plants or are reduced to single leaves (Fig. (Fig.11G) or to filamentous structures (14). In addition, bl and to mutants have a tendency to terminate shoot growth after formation of an inflorescence (Fig. (Fig.11E), a feature that was found to be very pronounced in bl-1. Furthermore, inflorescences of bl and to mutants are highly reduced, producing only 1 to 4 flowers per truss, compared with 7 to 12 in the wild type (Fig. (Fig.11 D and E). In addition, flowers tend to be fused and fasciated, with an increased number of flower organs. Floral organs with mixed character (sepal/petal) were sometimes observed (Fig. (Fig.11H).

Figure 1
Phenotypes of blind and torosa plants. Comparison of wild-type plants (A and D), bl-2 plants (B and G), to-1 plants (E and H), and plants expressing the RNAi construct GSMyb12 (C, F, and I). The pictures show stem segments with leaf axils (A–C ...

The recessive mutants bl-1, bl-2, and to-1 were crossed to each other and also to lateral suppressor (ls-1). F1 and F2 plants from crosses between bl-1, bl-2, and to-1 showed reduced side shoot formation and mutant inflorescences, supporting the conclusion that they are allelic. Crosses with ls resulted in wild-type F1 plants, and in the F2 generation the parental phenotypes segregated independently. ls, bl double mutants showed the typical morphological defects of both single mutants. As the bl and to mutants show similarity to the self pruning (sp) mutant (38) with respect to their precocious shoot termination, we have tested for the presence of a mutant sp allele. Crosses of bl-2 and to-1 to sp resulted in wild-type F1 plants, whereas the F1 progeny of bl-1 and sp showed an sp termination phenotype and normal side shoot formation. Molecular analysis of the bl and to mutants by using an sp-specific CAPS marker (38) revealed that only bl-1 contains a mutant sp allele (data not shown). These experiments demonstrated that bl-1, bl-2, and to-1 are allelic to each other and that the bl-1 strain is in addition homozygous for the mutant sp allele.

Integration of the Blind Gene into the Tomato RFLP Map.

Based on the segregation of morphological markers, the blind locus was previously mapped to the short arm of tomato chromosome 11 (39). To integrate bl into the tomato RFLP map, we have used a population of 121 F2 plants, obtained from an interspecific cross between L. esculentum (bl-2/bl-2) and L. pennellii (Bl/Bl). In this population, the mutant phenotype cosegregated with the L. esculentum allele of RFLP marker TG546. The two flanking markers, TG36 and TG46, map at a distance of 4.6 and 6.4 cM from bl, respectively.

The RFLP markers TG46, TG546, and TG36 were converted into CAPS markers and used to isolate informative recombinants from an additional 797 plants of the F2 population. The bl phenotype cosegregated with RFLP marker TG546, except for two plants in which it was linked to marker TG46 (Fig. (Fig.22A). TG46 and TG36 were separated from blind by 31 and 43 recombinations, respectively. These data place the blind locus between TG46 and TG546 at a distance of less than 0.3 cM from marker TG546.

Establishment of a YAC and Cosmid Contig.

Using primers specific for TG546, two overlapping YACs (YAC154, YAC293) were isolated from a genomic tomato YAC library (30). Additional clones were isolated by using RFLP marker TG46 (YAC321, YAC347) and the left end of the insert of YAC154 (YAC96, YAC233, YAC313). YACs 96 and 313 were shown by Southern analysis to hybridize to YAC347 and YAC154, demonstrating the establishment of a contiguous stretch of genomic DNA in the Bl region (Fig. (Fig.22A).

Polymorphic YAC end probes were used for RFLP analysis within the population of recombinant plants. Whereas the left end of YAC293 (YAC293L) and the right end of YAC154 (YAC154R) map between RFLP markers TG546 and TG36, the end probes YAC154L, YAC313L, YAC293R, YAC96L, and YAC233L co-map with TG546. Probes YAC321L and YAC96R dissect the TG546-TG46 interval into three pieces of similar size, with bl positioned on YAC96. CAPS marker analyses placed YAC233R between TG546 and bl, separated by one recombination breakpoint each, and YAC347R to the same position as bl. These data position the blind locus to YACs 313 and 96, close to YAC347R (Fig. (Fig.22A).

Using YAC347R and YAC233R, cosmid clones containing genomic DNA from wild-type tomato plants (Cos-clones; ref. 16) and plasmid clones containing restriction fragments derived from YAC313 (P-clones) were isolated (Fig. (Fig.22B). Sequences obtained from the ends of these clones were used to establish a contig of about 116 kb spanning the blind locus. This contig was sequenced, and several PCR markers derived from this sequence were mapped relative to bl. The PCR markers cos76E and FP2, separated by about 90 kb, delimit the region cosegregating with bl. Screening protein and nucleotide databases with overlapping subfragments of this region with the help of the BLAST algorithm (36) led to the identification of nine candidate genes (Table (Table1,1, Fig. Fig.22B).

Table 1
Putative genes in the Blind region

Identification and Characterization of the Blind Gene.

To identify the Blind gene, the mutant bl and to alleles were screened for sequence alterations. PCR products derived from four candidate genes possibly involved in transcriptional regulation (D), signal transduction (H and I), or plant hormone biosynthesis (F) were sequenced. In a total of 28 kb analyzed, only three sequence deviations between the wild-type and the three mutant alleles were detected; short deletions of 37 bp (position 427–463 on the cDNA; accession no. AF426174), 34 bp (position 395–428), and 1 bp (position 820) were found in candidate gene D of bl-1, bl-2, and to-1, respectively. to-2 was found to harbor the same 34-bp deletion as bl-2, confirming that both mutants are identical. Because all three deletions lead to frame shifts and very likely to a loss of protein function, we concluded that candidate D had a very high probability to represent the Blind gene.

The ORF of the putative Bl gene was amplified from flower bud RNA by RT-PCR. To determine the 5′ end of the transcript, independent products obtained in 5′ RACE experiments were sequenced. The two longest products started at the same base pair (position 1 of the cDNA), suggesting that this position corresponds to the 5′ end of the transcript. As determined by 3′ RACE experiments, the Bl transcript contains 147 bases of untranslated 3′ sequences. From these experiments, we conclude that the transcript has a length of 1,152 bp. To determine Bl transcript levels, RT-PCR analysis was performed with total RNA from different plant organs. Bl transcript levels were found to be highest in vegetative shoot tips, young flower buds, and axillary buds, low in roots, and very low in leaves (Fig. (Fig.3).3). The transcript could not be detected in stem tissue, even with an increased number of PCR cycles. The RNA was also detected in flower buds of bl and to mutants. Transcript levels in bl-1 and bl-2, but not in to-1, are reduced in comparison to wild type. Alignment of the cDNA and the corresponding genomic DNA revealed that the Bl coding region is interrupted by two introns (Fig. (Fig.44A). The first ATG of the transcript (position 61), which is preceded by stop codons in all three frames, initiates an ORF with a coding capacity for 315 aa.

Figure 3
Detection of Blind mRNA. Total RNA was extracted from different plant organs of the wild-type or from flower buds of the mutants and analyzed by RT-PCR. Amplification of actin cDNA was used to ensure that approximately equal amounts of cDNA were added ...
Figure 4
Blind is an R2R3 type Myb gene. (A) Exon–intron structure of the Blind gene. The transcript is represented as a bold line, the coding region as boxes, and the MYB domain is shown in gray. Arrows indicate the position of deletions in the different ...

RNAi Inhibition of Blind Function.

To obtain additional proof that the isolated Myb gene corresponds to Blind, a Bl RNAi (32) construct (pGSMyb12) was introduced into wild-type tomato plants. For this purpose, part of the candidate gene D ORF was expressed under the control of the cauliflower mosaic virus 35S promoter, with 531 bp of the RNA inserted in antisense orientation. Of 19 independent transgenic plants, 17 clearly showed morphological characteristics also observed in bl and to mutants (Fig. (Fig.11 C, F, and I). All of these plants were characterized by a reduction in the number of lateral shoots and the number of flowers per inflorescence. In addition, most plants showed a premature termination of the main axis and sometimes transformation of side shoots into leaves or leaf-like structures. Fused flowers were observed in two of the transgenic plants. In summary, most of the pGSMyb12-containing plants displayed a phenotype that was an almost perfect phenocopy of the bl and to mutants. In the progenies of five independent transformants, the phenocopy phenotype cosegregated with the presence of a T-DNA.

Blind Is a Member of the Myb Gene Family.

Comparison of the Bl protein sequence to the databases revealed considerable sequence similarity to members of the R2R3 class of MYB transcription factors. The predicted protein contains a DNA binding domain that is characteristic for a subgroup of the R2R3 class Myb genes. In A. thaliana, six genes of this subgroup (AtMyb36, AtMyb37, AtMyb38, AtMyb68, AtMyb84, and AtMyb87) have been identified (40), which show 76–86% identity within the first 118 aa (Fig. (Fig.44B). Other members of the R2R3 class Myb genes of Arabidopsis display only between 40% and 67% identical residues in this domain. In tomato, this R2R3 subgroup consists of at least five genes as indicated by the identification of expressed sequence tags from four additional transcripts. Furthermore, expressed sequence tags have been identified from Medicago trunculata, soybean, rice, wheat, and Sorghum.

The C-terminal domain of the Bl protein does not show obvious similarity to any other known protein sequence. It is rich in hydrophilic amino acids, especially in asparagines (21%), partly organized in homopolymeric stretches, a feature often found in activation domains of transcription factors. The frame shifts found in the three mutant alleles lead to an introduction of stretches of hydrophobic residues in this hydrophilic region. Deletions in the bl-1 and bl-2 alleles result in proteins altered after amino acids 123 and 112, respectively. Both mutant alleles may have the capacity to encode polypeptides of 173 aa and 174 aa, respectively, with C-terminal domains rich in leucine and isoleucine. to-1 harbors a single base-pair deletion leading to a change of the ORF after amino acid 253. The putative aberrant protein of 309 aa has a very hydrophobic C terminus rich in the amino acids leucine, isoleucine, and valine.

Discussion

Blind Is a Myb Gene of the R2R3 Class of Transcription Factors.

RFLP analysis placed the blind locus controlling the initiation of lateral meristems to a position between the RFLP markers TG36 and TG46 in close proximity to TG546 on the short arm of chromosome 11, confirming the mapping data obtained by classical crossing experiments (39). After establishment of a YAC and a cosmid/plasmid contig, the region cosegregating with the bl phenotype was narrowed down to a segment of about 90 kb. Within this region, nine putative genes have been identified. The extremely low level of DNA polymorphisms observed between different L. esculentum cultivars allowed us to identify the Bl coding region by sequence comparisons of wild-type and mutant alleles. The three mutant alleles are characterized by small deletions, leading to frame shifts in the third exon each, in a Myb gene of the R2R3 class, which is preferentially expressed in young developing tissues. Additional evidence for the identity of the Bl gene was obtained by an RNA interference experiment (32); more than 80% of transgenic plants expressing a Blind RNAi construct had a bl phenotype. The results of the mapping experiments, the sequence analyses, and the RNAi experiment clearly demonstrate that the isolated Myb gene is responsible, in its mutated form, for the bl phenotype.

Whereas the Blind MYB domain of 118 aa reveals similarity to other members of the R2R3 class MYB proteins, the C-terminal domain of the protein has no obvious sequence conservation to other known genes, a feature found for most of the R2R3 MYB genes (41). It is characterized by stretches of asparagine residues, resembling stretches of hydrophilic and acidic residues found in the activation domains of other transcription factors. In the three mutant alleles, this putative transcriptional activation domain is altered because of frame shifts, introducing, in each case, a hydrophobic terminus into the protein.

The Blind protein belongs to a subgroup within the family of R2R3 MYB proteins that is characterized by strong sequence conservation within the MYB domain. This subgroup consists of six members in the model species A. thaliana and of at least five members in tomato. R2R3 MYB proteins are involved in the regulation of very different pathways, but members of specific subgroups perform a much narrower spectrum of functions and may have some degree of redundancy (40). Recently, it has been shown that the two related MYB genes WEREWOLF and GLABRA1, controlling root hair development and shoot hair formation, respectively, encode proteins that can substitute each other. The different functions of these two regulators are specified by their expression patterns (42). Accordingly, we assume that Bl may be the founding member of a subfamily of genes with common regulatory functions. As the whole subgroup of six genes is known in A. thaliana, knock out mutants will be instrumental to test this assumption.

Blind Is a General Regulator of Shoot Branching in Tomato.

In tomato, the bl mutations interfere with the formation of all lateral shoot and inflorescence meristems, whereas the initiation and function of the primary SAM are unaffected. Vegetative side shoots, including sympodial shoots, are often missing, resulting in plants with a low number of branches, frequently terminating in an inflorescence. The failure to initiate new lateral meristems during reproductive development often results in inflorescences consisting of a single flower, which is the terminal structure of the main tomato shoot formed by the primary SAM. Alternatively, the inflorescences are made up of few, irregularly spaced flowers. In the tomato inflorescence, incomplete separation of the floral and the inflorescence meristems, which arise as a result of branching, may be the cause for the typical fused flowers of bl mutants.

The fact that the formation of lateral meristems is not completely inhibited may be explained in different ways. It is conceivable that other members of the Blind Myb subfamily are able to substitute to some extent for the missing function in bl mutants. Alternatively, the three bl alleles may have residual activity that is not sufficient to fully promote side shoot development.

Lateral Meristem Formation Is Controlled by Different Regulatory Pathways.

The three known regulators of lateral meristem formation, Ls, REV, and Bl, all belong to different families of transcription factors. Ls is a member of the GRAS protein family (4, 16), REV belongs to the HD-ZIP-START subgroup of homeodomain/leucine zipper transcription factors (10, 21, 22), and Bl is a member of the R2R3 class of MYB proteins. We do not know how the activities of these transcriptional regulators are integrated in a network of genetic control. The phenotypes of the respective mutants indicate only a partial overlap of the regulatory pathways in which these genes are involved. In ls mutants, axillary meristem formation is completely blocked during vegetative development, whereas development of the two side shoots preceding an inflorescence and inflorescence branching are only weakly affected. On the other hand, bl and rev mutants show defects in all types of lateral meristems, although lateral meristem formation is not completely blocked in any position of the plant body. Furthermore, Ls, Bl, and REV are involved in the control of developmental pathways, which are not directly linked to lateral meristem formation. Whereas in ls, where petal formation is almost completely suppressed (16), rev mutants show a defect in the formation of interfascicular fibers and secondary xylem (22). In bl mutants, the identity of first whorl flower organs tends to be modified. The different spectra of mutant phenotypes indicate that the activities of these three genes are not related to each other in a simple hierarchical order but that they are rather involved in independent pathways with an overlap in the control of axillary meristem initiation. This assumption could be tested by combining ls and bl alleles in one plant. ls, bl double mutants displayed a phenotype that was a combination of both single mutant phenotypes, strongly suggesting that the two genes act in independent pathways to promote lateral meristem formation. However, it cannot be excluded that both gene products interact during formation of lateral meristems. Interactions between Bl and REV as well as between Ls and REV will be analyzed when mutations in the respective genes can be combined in one species, e.g., in Arabidopsis. In addition, it will be important to establish relationships between these genes controlling lateral meristem initiation and other meristem regulators, like SHOOT MERSISTEMLESS (STM) and WUSCHEL (WUS). Preliminary evidence suggests that REV acts upstream of STM and WUS (10), as well as Ls acts upstream of STM (unpublished results) to activate their expression.

Acknowledgments

We thank the Tomato Genetic Resource Center (Davis, CA) and the Institute for Plant Genetics and Crop Plant Research (Gatersleben, Germany) for providing seed stocks. We are grateful to Dr. S. Tanksley for providing RFLP markers and to Dr. M. Ganal for making available the tomato YAC library. We thank Dr. K. Schumacher, Dr. R. Schmidt, and Dr. F. Salamini for critical reading of the manuscript and members of the laboratory for helpful discussions. This research was supported by grants from the Deutsche Forschungsgemeinschaft and the Società consortile Metapontum Agrobios a r. l. within the MEGA project granted by the Italian Ministero della Programmazione Economica e del Bilancio.

Abbreviations

SAM
shoot apical meristem
RFLP
restriction fragment length polymorphism
YAC
yeast artificial chromosome
CAPS
cleaved amplified polymorphic sequence
RT
reverse transcription
RACE
rapid amplification of cDNA ends
RNAi
RNA interference

Footnotes

This paper was submitted directly (Track II) to the PNAS office.

Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF426174).

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