A major challenge in biology is to understand how buds comprising a few cells can give rise to complex plant and animal appendages like leaves or limbs. We address this problem through a combination of time-lapse imaging, clonal analysis, and computational modeling. We arrive at a model that shows how leaf shape can arise through feedback between early patterns of oriented growth and tissue deformation. Experimental tests through partial leaf ablation support this model and allow reevaluation of previous experimental studies. Our model allows a range of observed leaf shapes to be generated and predicts observed clone patterns in different species. Thus, our experimentally validated model may underlie the development and evolution of diverse organ shapes.

In Arabidopsis, the basic leucine zipper transcription factor ABI5 activates several late embryogenesis-abundant genes, including AtEm1 and AtEm6 . However, the expression of many other seed maturation genes is independent of ABI5 . We investigated the possibility that ABI5 homologs also participate in the regulation of gene expression during seed maturation. We identified 13 ABI5 -related genes in the Arabidopsis genomic sequence. RNA gel blot analysis showed that seven of these genes are active during seed maturation and that they display distinct expression kinetics. We isolated and characterized two mutant alleles of one of these genes, AtbZIP12/EEL . Unlike abi5 , the eel mutations did not inhibit the expression of any of the maturation marker genes that we monitored. On the contrary, the accumulation of the AtEm1 and AtEm6 mRNAs was enhanced in eel mutant seeds compared with wild-type seeds. Gel mobility shift assays, combined with analysis of the genetic interactions among the eel and abi5 mutations, indicated that ABI5 and EEL compete for the same binding sites within the AtEm1 promoter. This study illustrates how two homologous transcription factors can play antagonistic roles to fine-tune gene expression.

The Arabidopsis abscisic acid (ABA) insensitive (ABI)5 transcription factor participates in the ABA-dependent induction of late embryogenesis abundant (LEA) genes in the ¢nal stages of seed development. We tested whether the VP16 transcriptional activation domain is su⁄cient to provide ABI5 with the ability to activate the AtEm LEA genes in vegetative tissues. We took advantage of a new transgenic seed selection assay based on green £uorescent protein (GFP) £uorescence and found that VP16-ABI5 triggered growth retardation and ABAindependent induction of AtEm1 in seedlings. These results indicate that ABI5 activation potential is a limiting step and might be a target for ABA signaling. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

The Arabidopsis thaliana genome contains approximately 80 genes encoding basic leucine zipper transcription factors, divided into 11 groups. Abscisic Acid-Insensitive 5 (ABI5) is one of the 13 members of group A and is involved in ABA signalling during seed maturation, and germination. Seven other members of this group are expressed during seed maturation, but only one of them (Enhanced Em Level, EEL) has been functionally characterized during this developmental phase. Since EEL and two other group A genes, AtbZIP67 and AREB3 (ABA-Responsive Element Binding protein 3), display similar mRNA temporal expression in whole siliques, it is suspected that they might share some overlapping functions. To address this question, the proteins' tissular and subcellular localization in transgenic Arabidopsis were precisely characterized, using translational fusions with a green fluorescent protein (GFP) expressed under the corresponding bZIP promoter. It was found that the three fusion proteins were expressed with a largely overlapping pattern and constitutively localized in the nuclei. An RNA interference approach (RNAi) was then used to knock out the expression of all three genes simultaneously. Endogenous EEL, AREB3, and AtbZIP67 transcripts could be specifically reduced, but no visible defects could be observed during seed maturation.

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A key approach to understanding how genes control growth and form is to analyze mutants in which shape and size have been perturbed. Although many mutants of this kind have been described in plants and animals, a general quantitative framework for describing them has yet to be established. Here we describe an approach based on Principal Component Analysis of organ landmarks and outlines. Applying this method to a collection of leaf shape mutants in Arabidopsis and Antirrhinum allows low-dimensional spaces to be constructed that capture the key variations in shape and size. Mutant phenotypes can be represented as vectors in these allometric spaces, allowing additive gene interactions to be readily described. The principal axis of each allometric space reflects size variation and an associated shape change. The shape change is similar to that observed during the later stages of normal development, suggesting that many phenotypic differences involve modulations in the timing of growth arrest. Comparison between allometric mutant spaces from different species reveals a similar range of phenotypic possibilities. The spaces therefore provide a general quantitative framework for exploring and comparing the development and evolution of form

SUMMARYLegumes have evolved the capacity to form a root nodule symbiosis with soil bacteria called rhizobia. The establishment of this symbiosis involves specific developmental events occurring both in the root epidermis (notably bacterial entry) and at a distance in the underlying root cortical cells (notably cell divisions leading to nodule organogenesis). The processes of bacterial entry and nodule organogenesis are tightly linked and both depend on rhizobial production of lipo-chitooligosaccharide molecules called Nod factors. However, how these events are coordinated remains poorly understood. Here, we have addressed the roles of two key symbiotic genes of Medicago truncatula, the lysin motif (LysM) domain-receptor like kinase gene NFP and the calcium-and calmodulindependent protein kinase gene DMI3, in the control of both nodule organogenesis and bacterial entry. By complementing mutant plants with corresponding genes expressed either in the epidermis or in the cortex, we have shown that epidermal DMI3, but not NFP, is sufficient for infection thread formation in root hairs. Epidermal NFP is sufficient to induce cortical cell divisions leading to nodule primordia formation, whereas DMI3 is required in both cell layers for these processes. Our results therefore suggest that a signal, produced in the epidermis under the control of NFP and DMI3, is responsible for activating DMI3 in the cortex to trigger nodule organogenesis. We integrate these data to propose a new model for epidermal/cortical crosstalk during early steps of nodulation.

SummaryMyc-LCOs are newly identified symbiotic signals produced by arbuscular mycorrhizal (AM) fungi. Like rhizobial Nod factors, they are lipo-chitooligosaccharides that activate the common symbiotic signalling pathway (CSSP) in plants. To increase our limited understanding of the roles of Myc-LCOs we aimed to analyse Myc-LCO-induced transcriptional changes and their genetic control.Whole genome RNA sequencing (RNA-seq) was performed on roots of Medicago truncatula wild-type plants, and dmi3 and nsp1 symbiotic mutants affected in nodulation and mycorrhizal signalling. Plants were treated separately with the two major types of Myc-LCOs, sulphated and nonsulphated.Generalized linear model analysis identified 2201 differentially expressed genes and classified them according to genotype and/or treatment effects. Three genetic pathways for Myc-LCO-regulation of transcriptomic reprogramming were highlighted: DMI3-and NSP1-dependent; DMI3-dependent and NSP1-independent; and DMI3-and NSP1-independent. Comprehensive analysis revealed overlaps with previous AM studies, and highlighted certain functions, especially signalling components and transcription factors.These data provide new insights into mycorrhizal signalling mechanisms, supporting a role for NSP1, and specialisation for NSP1-dependent and -independent pathways downstream of DMI3. Our data also indicate significant Myc-LCO-activated signalling upstream of DMI3 and/or parallel to the CSSP and some constitutive activity of the CSSP.