The asymmetric leaves2 (as2) mutant of Arabidopsis thaliana generated leaf lobes and leaflet-like structures from the petioles of leaves in a bilaterally asymmetric manner.  Both the delayed formation of the primary vein and the asymmetric formation of secondary veins were apparent in leaf primordia of as2 plants.  A distinct midvein, which is the thickest vein and is located in the longitudinal center of the leaf lamina of wild-type plants, was often rudimentary even in mature as2 leaves.  However, several parallel veins of very similar thickness were evident in such leaves.  The complexity of venation patterns in all leaf-like organs of as2 plants was reduced.  The malformed veins were visible prior to the development of asymmetry of the leaf lamina and were maintained in mature as2 leaves.  Culture in vitro on phytohormone-free medium of leaf sections from as2 mutants and from the asymmetric leaves1 (as1) mutant, which has a phenotype similar to that of as2, revealed an elevated potential in both cases for regeneration of shoots from leaf cells.  Analysis by the reverse transcription-polymerase chain reaction showed that transcripts of the KNAT1, KNAT2 and KNAT6 (a recently identified member of the class 1 knox family) genes accumulated in the leaves of both as2 and as1 plants but not in wild-type leaves.  Transcripts of the STM gene also accumulated in as1 leaves.  These findings suggest that, in leaves, the AS2 and AS1 genes repress the expression of these homeobox genes, which are thought to maintain the indeterminate cell state in the shoot apical meristem.  We propose that AS2 and AS1 might be involved in establishment of a prominent midvein and of networks of other veins as well as in the formation of the symmetric leaf lamina, which might be related to repression of expression of class 1 knox homeobox genes in leaves.
 We have characterized the AS2 gene, which appears to encode a novel protein with cysteine repeats (designated the C-motif) and a leucine-zipper-like sequence in the amino-terminal half of the primary sequence.  The Arabidopsis genome contains 42 putative genes that potentially encode proteins with conserved amino acid sequences that include the C-motif and the leucine-zipper-like sequence in the amino-terminal half.  Thus, the AS2 protein belongs to a novel family of proteins that we have designated the AS2 family.  Members of this family except AS2 also have been designated ASLs (AS2-like proteins).  Transcripts of AS2 were detected mainly in adaxial domains of cotyledonary primordia.  Green fluorescent protein-fused AS2 was concentrated in plant cell nuclei.  Overexpression of AS2 cDNA in transgenic Arabidopsis plants resulted in upwardly curled leaves, which differed markedly from the downwardly curled leaves generated by loss-of-function mutation of AS2.  Our results suggest that AS2 functions in the transcription of a certain gene(s) in plant nuclei and thereby controls the formation of a symmetric flat leaf lamina and the establishment of a prominent midvein and other patterns of venation.
 

Figure (A - D) Characteristic features of leaves of the asymmetric leaves2 (as2) mutant.  A, leaf shape of as2; B, leaf shape of wild type; C, venation pattern of as2; D, venation pattern of wild type.  (E and F) The generation of shoots on as2 leaves in vitro.   Sections of the as2 (E) and wild-type (F) leaves were photographed after incubation for 16 days on hormone-free MS medium.  Several percent of leaf sections from as2 have produced shoots at the sinus of a leaf lobe while no section from the wild-type leaf have generated shoots but roots at the efficiency of 12% (see Semiarti et al., 2001).  (G) Domain organization and characteristic features of the predicted AS2 protein.  The region indicated by a bracket below the box is the AS2 domain.  The shaded box and the striped box represent the C-motif and the leucine-zipper like sequence, respectively.  Sites of as2-1, as2-2, as2-4, and as2-5mutations are indicated.  Numbers below the box indicate positions of amino acid residues.