Of blades and branches: understanding and expanding the Arabidopsis ad/abaxial regulatory network through target gene identification.

HD-ZIPIII and KANADI transcription factors have opposing and dramatic affects on plant development. Analysis of mutants shows these proteins to be master regulators of ad/abaxial (i.e., upper/lower) leaf polarity, leaf blade outgrowth, and branch formation. Because these factors do their work by regulating other genes, we have focused our attention on defining their targets. We have found overlap between the ad/abaxial regulatory pathway and hormone signaling pathways, especially pathways of abscisic acid and auxin signaling. This has led to the discovery that abscisic acid signaling acts upstream of HD-ZIPIII and KANADI in the control of germination and may ultimatelyexplain howenvironmental stress pathways control new growth at the shoot apex. Auxin signaling conversely is downstream from HD-ZIPIII and KANADI action with these factors controlling targets at all steps of auxin action—biosynthesis, transport, regulation of transport, and signaling. Based on thesefindings,weproposeamodelinwhichtheHD-ZIPIIIandKANADIfactorspatternauxinresponseintheembryo.Finally, many genes targeted for control by HD-ZIPIII and KANADI proteins are themselves transcription factors—indicating these master regulators call up tissue specific subprograms of transcriptional control to affect the many polar differences observed across tissues. Leaves are typically thin, flat organs specialized for harvesting light energy from the sun. Using this energy, in what must have seemed to early scientists like a feat startlingly close to alchemy, leaves turn carbon dioxide and water into sugar and oxygen. The flattened nature of the leaf, which maximizes surface area both for light capture and for gas exchange, exists in tension with the need to prevent excessive water loss, especially in dry environments. Thus, the size and shape of the leaf blade variesgreatlyamong species and with the diversehabitats found on land. Plants growing in dry environments often make narrow leaves with limited surface area that are sufficient to support the slow growth allowed when water is limiting. On the other hand, plants growing in moist, nutrient-rich environments may make large, tender leaves the better to compete with their neighbors for light and space. Leaf shape may even vary substantially during the lifespan of an individual plant, especially when the environment changes substantially during this time. Extreme examples of this are aquatic plants in which the early leaves formed under water have very different shapes from the later leaves formed above water. We have been studying the mechanism of action of regulators of ad/abaxial leaf polarity in the plant. Besides directing different patterns of cellular differentiation in the upper and lower halves of the leaf, these regulators also control blade outgrowth and the formation of new branches. Owing to their ability to influence such basic growth properties, the ad/abaxial regulators are expected to be perfectly suited targets of evolutionary modifications that allow plants to survive in many environments. Their activity may also be affected by physiological signals reporting on environmental change occurring during the course of the lifetime of a single plant. As these regulators are transcription factors, the key to understanding how they work must rest, at least in part, in an understanding of what genes they control. Our approach has been to identify, with as much confidence as possible, the genes controlled by the ad/abaxial regulators and to use this information to identify newactors required for leaf development and to test new hypotheses. In this paper, we summarize the discoveries and the hypotheses that have emerged from these studies.