To understand the role the homeobox gene knotted1 (kn1) plays in development, we undertook to identify its downstream targets. A subtractive hybridization scheme to isolate up-regulated target genes was devised, taking advantage of the pattern of expression of kn1 in the dominant Kn1-N2 allele. In this allele, kn1 is ectopically expressed in localized regions of the leaf, usually close to veins, whereas in wild-type plants kn1 expression is undetectable in leaves. mRNA from unexpanded leaves of 10-day old Kn1-N2 seedlings was isolated and converted into double-stranded cDNA. Some of this double-stranded cDNA was used to construct a cDNA library. First strand wild type leaf cDNA was synthesized on mRNA attached to magnetic beads and the resulting RNA:DNA hybrids denatured to remove the RNA strand. A large excess of wild type cDNA attached to the beads was then hybridized to a trace amount of denatured double-stranded cDNA made from Kn1 leaves. After exhaustive hybridization the renatured cDNA left in solution was used to make a cDNA library, the wild-type cDNA population being removed along with the beads. The cDNAs in solution represented unique clones present in the Kn1 leaf cDNA population. The subtracted library was screened with a subtracted probe, and in addition, fifty random clones were picked and analyzed.

A total of seven different genes were obtained from the two approaches. From the library screen, ABA-inducible glycine-rich protein (GRP) and CHEM2, a stress-inducible GRP, were obtained. The ABA-inducible GRP, also known as MA16, contains a consensus RNA-binding motif. From the randomly picked clones, five additional groups of cDNAs were isolated, each group comprising 1 to 2 clones. These included c87, a cDNA showing some homology to plant S-like ribonucleases, a cDNA showing homology to BnC24, a Brassica napus gene homologous to a human tumour gene, breast basic conserved1 (bbc1), and 3 cDNA fragments which showed no significant homology to any sequences in the GenBank database. The two GRPs were also represented in the randomly picked clones.

On Northern blots, c87 showed a substantial increase in expression in Kn1 leaves over wild type leaves (Figure 1A), showing that the subtraction protocol enriched for cDNAs that are more abundant in Kn1 leaves. ABA-inducible GRP and CHEM2 also showed increases in expression in Kn1 leaf tissue, while the other genes did not show an increase or were not detectable on RNA blots. In addition, c87 showed increased mRNA levels in leaves of transgenic maize plants constitutively expressing kn1 (Figure 1B). Approximately 3 kb of c87 genomic sequence upstream of the transcription start site was obtained. A KN1 homeodomain peptide bound with low affinity to fragments of the c87 promoter in gel retardation assays. Full-length cDNAs for c87 were obtained and used to generate probes for in situ hybridization on tissue from wild-type and Kn1 seedlings. c87 mRNA was detected in Kn1 leaves but not in wild-type leaves. Localized expression of c87 was detected in ears, in structures closely associated with stamen primordia, possibly lodicules. In tassels, c87 is localized to the L1 and L2 cell layers on the abaxial side of developing flowers. Later in development c87 expression appears as a ring at the base of the growing point of each floret. Determination of the map location of c87 is in progress.

Figure 1. Expression levels of c87 mRNA in wild-type, Kn1 and 35S::kn1 leaves. A. Northern blot showing the increase in expression of c87 in Kn1 leaves as compared to wild-type leaves. Expression is also detectable in meristem enriched tissue. 10 µg of total RNA was loaded in each lane. The blot was reprobed with a maize ubiqutin (ubi) probe as a loading control. B. Northern blot of leaf RNA from wild-type, Kn1 and 35S::kn1 plants probed with a c87 cDNA probe. Kn1 and 35S::kn1 (kn trans) leaves show increased levels of c87 mRNA. 10 µg of total RNA was loaded in each lane. The blot was reprobed with a maize tubulin probe as a loading control.

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