Getting in Shape? Leaves Work It Out with KANADI1

Maybe your New Year’s resolution involved getting in shape—that’s something developing leaves do all the time. Instead of working on their abs and glutes, however, leaves specify adaxial (upper) and abaxial (lower) cell fates (reviewed in Yamaguchi et al., 2012). This polarity initiates from signals within the shoot apical meristem, which cause the two sides of the leaf to develop different cell types. These different cell types form a boundary that promotes growth of the leaf blade, producing a large surface area that allows the leaf to intercept the maximum amount of light and thus conduct photosynthesis most efficiently. In addition to promoting leaf growth at their junction, abaxial and adaxial leaf surfaces also develop adaptive morphological differences to reduce water loss, discourage herbivory, and increase photosynthetic efficiency. For example, the adaxial surface may have a thicker epidermis and denser mesophyll cells, but the abaxial surface may have more stomata.

Opposing actions aren’t just for the weight room either—determining abaxial and adaxial cell fate requires the mutually antagonistic actions of different signals and multiple transcription factors. On the upper side of the leaf, class III HOMEODOMAIN–LEUCINE ZIPPER (HD-ZIPIII) transcription factors promote adaxial cell fate. On the lower side, KANADI (KAN) transcription factors and AUXIN RESPONSE FACTORs promote abaxial cell fate. To get at the mechanism behind these interactions, Huang et al. (pages 246–262) examine the function of KAN1. Starting with a mutant that affects regulation of a KAN1-dependent gene, and refining their findings with oligonucleotide selection experiments, the authors find that KAN1 binds to a CNATA(A/T) motif. Moreover, this motif confers KAN1-dependent repression on a reporter gene (see figure) expressed under the control of the GAL4 upstream activating sequence.

Complementary expression of KAN1 and a reporter regulated by the KAN1 cis-element. KAN1 binds a specific cis-element, the CNATA(A/T) motif; a line with this element upstream of β-glucuronidase (GUS) shows GUS staining on the adaxial side of developing leaves (left). By contrast, GUS expression under the control of the KAN1 promoter shows GUS staining in the shoot apical meristem, leaf primordia, and the abaxial side of the leaf. (Reprinted from Huang et al. [2014], Figure 2.)

The authors then identified KAN1 target genes. Ectopic expression of KAN1 arrests seedling development; to circumvent this problem, the authors produced a dexamethasone-inducible KAN1 expression construct. Inducible expression of KAN1 causes a much milder phenotype (shorter petioles and leaf curling) than constitutive expression. This allowed the authors to conduct microarray analysis, which identified more than 200 genes up- or downregulated in the transgenic plants ectopically expressing KAN1. To identify direct targets, the authors used chromatin immunoprecipitation, finding that KAN1 directly targets most of the downregulated genes, but only some of the upregulated genes, indicating that it represses transcription. KAN1 binds to many, but not all, of the CNATA(A/T) motifs, indicating that other factors contribute to KAN1 binding, possibly including the corepressor TOPLESS. The direct targets of KAN1 include transcription factors and many genes involved in auxin signaling and transport. Moreover, comparison to other transcriptome studies revealed that KAN1 and the HD-ZIP transcription factor REVOLUTA regulate the expression of some genes in the same families but produce opposite effects, possibly resulting in differential expression of auxin transport proteins in cells expressing KAN1 or REVOLUTA. The balance of transcription factors and the resulting auxin gradients provide a key means for the leaf to get in shape, but figuring out their precise mechanisms will also give researchers plenty of mental workouts for the coming year.