Size regulation of the lateral organ initiation zone and its role in determining cotyledon number in conifers.

Introduction: Unlike monocots and dicots, many conifers, particularly Pinaceae, form three or more cotyledons. These are arranged in a whorl, or ring, at a particular distance from the embryo tip, with cotyledons evenly spaced within the ring. The number of cotyledons, nc, varies substantially within species, both in clonal cultures and in seed embryos. nc variability reflects embryo size variability, with larger diameter embryos having higher nc. Correcting for growth during embryo development, we extract values for the whorl radius at each nc. This radius, corresponding to the spatial pattern of cotyledon differentiation factors, varies over three-fold for the naturally observed range of nc. The current work focuses on factors in the patterning mechanism that could produce such a broad variability in whorl radius. Molecularly, work in Arabidopsis has shown that the initiation zone for leaf primordia occurs at a minimum between inhibitor zones of HD-ZIP III at the shoot apical meristem (SAM) tip and KANADI (KAN) encircling this farther from the tip. PIN1-auxin dynamics within this uninhibited ring form auxin maxima, specifying primordia initiation sites. A similar mechanism is indicated in conifer embryos by effects on cotyledon formation with overexpression of HD-ZIP III inhibitors and by interference with PIN1-auxin patterning. Methods: We develop a mathematical model for HD-ZIP III/KAN spatial localization and use this to characterize the molecular regulation that could generate (a) the three-fold whorl radius variation (and associated nc variability) observed in conifer cotyledon development, and (b) the HD-ZIP III and KAN shifts induced experimentally in conifer embryos and in Arabidopsis. Results: This quantitative framework indicates the sensitivity of mechanism components for positioning lateral organs closer to or farther from the tip. Positional shifting is most readily driven by changes to the extent of upstream (meristematic) patterning and changes in HD-ZIP III/KAN mutual inhibition, and less efficiently driven by changes in upstream dosage or the activation of HD-ZIP III. Sharper expression boundaries can also be more resistant to shifting than shallower expression boundaries. Discussion: The strong variability seen in conifer nc (commonly from 2 to 10) may reflect a freer variation in regulatory interactions, whereas monocot (nc = 1) and dicot (nc = 2) development may require tighter control of such variation. These results provide direction for future quantitative experiments on the positional control of lateral organ initiation, and consequently on plant phyllotaxy and architecture.

Identification of genes expressed in vascular tissues using NPA-induced vascular overgrowth in Arabidopsis.

The genetic basis of vascular differentiation and function is relatively poorly understood, partly due to the difficulty of screening for mutants defective in internal vascular tissues. Here we present an approach based on a predicted increase in vascular-related gene expression in response to an auxin transport inhibitor-induced vascular overgrowth. We used microarray analyses to identify 336 genes that were up-regulated > or =2-fold in shoot tissues of Arabidopsis thaliana showing vascular overgrowth. Promoter-marker gene fusions revealed that 38 out of 40 genes with > or =4-fold up-regulation in vascular overgrowth tissues had vascular-related expression in transgenic Arabidopsis plants. Obtained expression patterns included cambial tissues and differentiating xylem, phloem and fibers. A total of 15 genes were found to have vascular-specific expression patterns in the leaves and/or inflorescence stems. This study provides empirical evidence of the efficiency of the approach and describes for the first time the in situ expression patterns of the majority of the assessed genes.

Dynamics of MONOPTEROS and PIN-FORMED1 expression during leaf vein pattern formation in Arabidopsis thaliana.

Genetic evidence links the Arabidopsis MONOPTEROS (MP) and PIN-FORMED1 (PIN1) genes to the patterning of leaf veins. To elucidate their potential functions and interactions in this process, we have assessed the dynamics of MP and PIN1 expression during vascular patterning in Arabidopsis leaf primordia. Both genes undergo a dynamic process of gradual refinement of expression into files one to two cells wide before overt vascular differentiation. The subcellular distribution of PIN1 is also gradually refined from a non-polar distribution in isodiametric cells to strongly polarized in elongated procambial cells and provides an indication of overall directions of auxin flow. We found evidence that MP expression can be activated by auxin exposure and that PIN1 as well as DR5::GUS expression is defective in mp mutant leaves. Taken together the results suggest a feedback regulatory loop that involves auxin, MP and PIN1 and provide novel experimental support for the canalization-of-auxin-flow hypothesis.