LETTERS TO THE EDITOR

INTERFASCICULAR FIBERLESS1 Is the Same Gene as REVOLUTA

The recently cloned INTERFASCICULAR FIBERLESS1 (IFL1) gene encodes a homeodomain–leucine zipper protein (HD-ZIP) that spatially regulates fiber differentiation in Arabidopsis (Zhong and Ye 1999 ). Mutations of the IFL1 gene are recessive and highly pleiotropic. In ifl1 mutants, normal interfascicular fibers are absent from the inflorescence stem and the differentiation of both xylary fibers and vessel elements in vascular bundles is disrupted. They further display long pendant stems, dark green leaves, delayed senescence, and fewer lateral branches (Zhong et al. 1997 ; Zhong and Ye 1999 ). These morphological characteristics are similar to those of plants with a defect in REVOLUTA (REV), a gene that influences aerial architecture by regulating the relative growth of apical versus non-apical meristems (Alvarez 1994 ; Talbert et al. 1995 ).

We recently discovered a putative homeobox gene, MUP24.4, within P1 clone MUP24 (GenBank accession number AB005246). Plants carrying a T-DNA insertion in the MUP24.4 sequence were then obtained by PCR-based screening of DNA pools from the Jack collection of insertional mutants (Campisi et al. 1999 ). The T-DNA insertion was located 466 bp downstream of the putative start codon, and was predicted to create a null mutation (Fig 1). Plants heterozygous for the T-DNA insertion appeared wild type, whereas homozygotes had a number of distinctive features reminiscent of the rev mutant. The most prominent characteristic was a failure in the development of all types of apical meristem: lateral shoot meristems in the axils of cauline and rosette leaves were often completely absent, or replaced by a solitary leaf. These effects were most evident in higher order shoots, but in some cases, the primary shoot meristem also failed, terminating growth in a cluster of filamentous structures. Compared to wild-type plants, the mutant showed a dramatic reduction in branching at maturity, delayed senescence, and enlarged revolute leaves. Defects in the floral meristem, moreover, resulted in enlarged floral organs, altered organ numbers, or the replacement of floral organs by filamentous structures.

View larger version (15K): [in this window] [in a new window]   Figure 1. Structure of the REVOLUTA Gene. The coding sequence between the start (ATG) and stop (TGA) codons is 4200 bp in length and consists of 18 exons and 17 introns. Exons are represented by boxes and introns by single lines. Arrows indicate the position of lesions that give rise to mutant alleles. Positions of the ifl1 alleles are taken from Zhong and Ye 1999 .

The similarity between the rev phenotype and that of the mup24.4 insertion mutant raised the possibility that the two genes were allelic. To test this hypothesis, we isolated the MUP24.4 sequence from rev-1 mutants and wild-type plants of ecotype Nossen (the background in which the rev-1 mutation had been isolated). The MUP24.4 sequence from rev-1 exhibited eight single-base changes compared to that from wild type Nossen (and 9 differences compared to wild type Columbia, reflecting a single base polymorphism between Nossen and Columbia in the 5th intron). Of these eight changes, one was upstream of the putative start codon, four were present in putative introns, and two were present in the 3' untranslated region. The eighth change was a G-to-A substitution predicted to disrupt the splice site at the junction between the eleventh intron and the twelfth exon (Fig 1), thereby implicating MUP24.4 as the REV gene. To confirm this possibility, homozygotes for the mup24.4 T-DNA insertion were crossed to rev-1 homozygotes: all F1 plants from this complementation test exhibited the rev phenotype.

The REV gene consists of 18 exons, which are predicted to encode an HD-ZIP protein of 842 amino acids, the sequence of which is identical to that of IFL1 (Zhong and Ye 1999 ). (We predict, however, that translation starts two codons prior to the ATG suggested by Zhong and Ye 1999 in their published protein sequence). Thus, the ifl1 mutations are, in fact, alleles of rev.

The finding that IFL1 is REV might help explain the deficiencies of fiber differentiation in the mutant. Lignified fiber cells are essential in providing support for the plant stem, and are thought to develop in response to the polar auxin flow that originates at the shoot tips (Aloni 1987 ). IFL1 was proposed to act either by regulating polar auxin flow or by regulating the genes involved in the transduction of hormonal signals that trigger fiber differentiation (Zhong and Ye 1999 ). REV is considered to be essential for apical meristem development. Since the auxin stream that induces fiber differentiation derives from shoots, it seems reasonable to suggest that defects in shoot meristem development would alter the polar auxin flow, and as a consequence, influence fiber differentiation.

The precise role of REV remains elusive. It has been suggested that REV promotes the growth of apical meristems (including floral meristems) at the expense of nonapical (cambial) meristems (Talbert et al. 1995 ). It is not yet clear, however, whether expression data support such a role. Strong expression of REV has been detected in interfascicular regions and developing vascular tissue, but in situ expression analysis of apical meristems has not yet been reported. REV is a group III HD-ZIP protein and shares high sequence similarity (and organization) with the proteins encoded by three other Arabidopsis genes: Athb8, Athb9, and Athb14 (Sessa et al. 1998 ). It is possible, therefore, that these genes act together in the same developmental process. In support of this suggestion, Athb8 shows an expression pattern similar to that of REV and is transcribed in the procambial regions of vascular bundles (Baima et al. 1995 ). Thus, to gain a full understanding of REV function and its contribution to plant architecture, it will be necessary to study the gene in conjunction with the other homologs.

	ACKNOWLEDGMENTS

We thank Marsha Pilgrim, Luc Adam, and Roderick Kumimoto for identification of the T-DNA insertion in MUP24.4.

	REFERENCES

TOP REFERENCES

Aloni, R. (1987) Differentiation of vascular tissues. Annu. Rev. Plant Physiol. 38:179-204.

Alvarez, J. (1994) The SPITZEN gene. In Bowman J., ed. Arabidopsis: An Atlas of Morphology and Development. New York, Springer-Verlag. 188–189.pp.

Baima, S., Nobili, F., Sessa, G., Lucchetti, S., Ruberti, I., and Morelli, G. (1995) The expression of the Athb-8 homeobox gene is restricted to provascular cells in Arabidopsis thaliana.. Development 12:4171-4182.

Campisi, L., Yang, Y., Yi, Y., Heilig, E., Herman, B., Cassista, A. J., Allen, D. W., Xiang, H., and Jack, T. (1999) Generation of enhancer trap lines in Arabidopsis and characterization of expression patterns in the inflorescence. Plant Journal 17:699-707.

Sessa, G., Steindler, C., Morelli, G., and Ruberti, I. (1998) The Arabidopsis Athb-8, -9 and -14 genes are members of a small gene family coding for highly related HD-ZIP proteins. Plant Mol. Biol. 38:609-622.

Talbert, P. B., Adler, H. T., Parks, D. W., and Comai, L. (1995) The REVOLUTA gene is necessary for limiting cell divisions in the leaves and stems of Arabidopsis thaliana.. Development 121:2723-2735.

Zhong, R., Jennifer, J. T., and Ye, Z.-H. (1997) Disruption of interfascicular fiber differentiation in an Arabidopsis mutant. Plant Cell 9:2159-2170.

Zhong, R., and Ye, Z.-H. (1999) IFL1, a gene regulating interfascicular fiber differentiation in Arabidopsis encodes a homeodomain-leucine zipper protein. Plant Cell 11:2139-2152.

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