Pradeep Das

Patterns of Auxin Transport and Gene Expression during Primordium Development Revealed by Live Imaging of the Arabidopsis Inflorescence Meristem

BACKGROUND Plants produce leaf and flower primordia from a specialized tissue called the shoot apical meristem (SAM). Genetic studies have identified a large number of genes that affect various aspects of primordium development including positioning, growth, and differentiation. So far, however, a detailed understanding of the spatio-temporal sequence of events leading to primordium development has not been established. RESULTS We use confocal imaging of green fluorescent protein (GFP) reporter genes in living plants to monitor the expression patterns of multiple proteins and genes involved in flower primordial developmental processes. By monitoring the expression and polarity of PINFORMED1 (PIN1), the auxin efflux facilitator, and the expression of the auxin-responsive reporter DR5, we reveal stereotypical PIN1 polarity changes which, together with auxin induction experiments, suggest that cycles of auxin build-up and depletion accompany, and may direct, different stages of primordium development. Imaging of multiple GFP-protein fusions shows that these dynamics also correlate with the specification of primordial boundary domains, organ polarity axes, and the sites of floral meristem initiation. CONCLUSIONS These results provide new insight into auxin transport dynamics during primordial positioning and suggest a role for auxin transport in influencing primordial cell type.

The auxin signalling network translates dynamic input into robust patterning at the shoot apex

The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large‐scale analysis of the Aux/IAA‐ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin‐based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA‐ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.

The homeotic protein AGAMOUS controls microsporogenesis by regulation of SPOROCYTELESS.

The Arabidopsis homeotic gene AGAMOUS (AG) is necessary for the specification of reproductive organs (stamens and carpels) during the early steps of flower development. AG encodes a transcription factor of the MADS-box family that is expressed in stamen and carpel primordia. At later stages of development, AG is expressed in distinct regions of the reproductive organs. This suggests that AG might function during the maturation of stamens and carpels, as well as in their early development. However, the developmental processes that AG might control during organogenesis and the genes that are regulated by this factor are largely unknown. Here we show that microsporogenesis, the process leading to pollen formation, is induced by AG through activation of the SPOROCYTELESS gene (SPL, also known as NOZZLE,NZZ), a regulator of sporogenesis. Furthermore, we demonstrate that SPL can induce microsporogenesis in the absence of AG function, suggesting that AG controls a specific process during organogenesis by activating another regulator that performs a subset of its functions.

Pattern formation during de novo assembly of the Arabidopsis shoot meristem.

Most multicellular organisms have a capacity to regenerate tissue after wounding. Few, however, have the ability to regenerate an entire new body from adult tissue. Induction of new shoot meristems from cultured root explants is a widely used, but poorly understood, process in which apical plant tissues are regenerated from adult somatic tissue through the de novo formation of shoot meristems. We characterize early patterning during de novo development of the Arabidopsis shoot meristem using fluorescent reporters of known gene and protein activities required for shoot meristem development and maintenance. We find that a small number of progenitor cells initiate development of new shoot meristems through stereotypical stages of reporter expression and activity of CUP-SHAPED COTYLEDON 2 (CUC2), WUSCHEL (WUS), PIN-FORMED 1 (PIN1), SHOOT-MERISTEMLESS (STM), FILAMENTOUS FLOWER (FIL, also known as AFO), REVOLUTA (REV), ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) and CLAVATA 3 (CLV3). Furthermore, we demonstrate a functional requirement for WUS activity during de novo shoot meristem initiation. We propose that de novo shoot meristem induction is an easily accessible system for the study of patterning and self-organization in the well-studied model organism Arabidopsis.

Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution

Quantitative information on growing organs is required to better understand morphogenesis in both plants and animals. However, detailed analyses of growth patterns at cellular resolution have remained elusive. We developed an approach, multiangle image acquisition, three-dimensional reconstruction and cell segmentation–automated lineage tracking (MARS-ALT), in which we imaged whole organs from multiple angles, computationally merged and segmented these images to provide accurate cell identification in three dimensions and automatically tracked cell lineages through multiple rounds of cell division during development. Using these methods, we quantitatively analyzed Arabidopsis thaliana flower development at cell resolution, which revealed differential growth patterns of key regions during early stages of floral morphogenesis. Lastly, using rice roots, we demonstrated that this approach is both generic and scalable.

Cytokinin signalling inhibitory fields provide robustness to phyllotaxis

How biological systems generate reproducible patterns with high precision is a central question in science. The shoot apical meristem (SAM), a specialized tissue producing plant aerial organs, is a developmental system of choice to address this question. Organs are periodically initiated at the SAM at specific spatial positions and this spatiotemporal pattern defines phyllotaxis. Accumulation of the plant hormone auxin triggers organ initiation, whereas auxin depletion around organs generates inhibitory fields that are thought to be sufficient to maintain these patterns and their dynamics. Here we show that another type of hormone-based inhibitory fields, generated directly downstream of auxin by intercellular movement of the cytokinin signalling inhibitor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), is involved in regulating phyllotactic patterns. We demonstrate that AHP6-based fields establish patterns of cytokinin signalling in the meristem that contribute to the robustness of phyllotaxis by imposing a temporal sequence on organ initiation. Our findings indicate that not one but two distinct hormone-based fields may be required for achieving temporal precision during formation of reiterative structures at the SAM, thus indicating an original mechanism for providing robustness to a dynamic developmental system.

The Role of Mechanical Forces in Plant Morphogenesis

The shape of an organism relies on a complex network of genetic regulations and on the homeostasis and distribution of growth factors. In parallel to the molecular control of growth, shape changes also involve major changes in structure, which by definition depend on the laws of mechanics. Thus, to understand morphogenesis, scientists have turned to interdisciplinary approaches associating biology and physics to investigate the contribution of mechanical forces in morphogenesis, sometimes re-examining theoretical concepts that were laid out by early physiologists. Major advances in the field have notably been possible thanks to the development of computer simulations and live quantitative imaging protocols in recent years. Here, we present the mechanical basis of shape changes in plants, focusing our discussion on undifferentiated tissues. How can growth be translated into a quantified geometrical output? What is the mechanical basis of cell and tissue growth? What is the contribution of mechanical forces in patterning?

TGF-β signaling, Smads, and tumor suppressors

The transforming growth factor‐β (TGF‐β) superfamily is used throughout animal development for regulating the growth and patterning of many tissue types. During the past few years, rapid progress has been made in deciphering how TGF‐β signals are transduced from outside the cell to the nucleus. This progress is based on biochemical studies in vertebrate systems and a combination of genetic studies in Drosophila and Caenorhabditis elegans. These studies have identified a novel family of signaling proteins, the Smad family. Smads can act positively and be phosphorylated by TGF‐β‐like receptors or can act negatively and prevent activation of the positively acting group. The positively acting Smads translocate to the nucleus, bind DNA, and act as transcriptional activators. Thus, genetic and biochemical studies suggest a very simple signaling pathway, in which Smads are the primary downstream participant. BioEssays 20:382–390, 1998.

Floral stem cell termination involves the direct regulation of AGAMOUS by PERIANTHIA

In Arabidopsis, the population of stem cells present in young flower buds is lost after the production of a fixed number of floral organs. The precisely timed repression of the stem cell identity gene WUSCHEL (WUS) by the floral homeotic protein AGAMOUS (AG) is a key part of this process. In this study, we report on the identification of a novel input into the process of floral stem cell regulation. We use genetics and chromatin immunoprecipitation assays to demonstrate that the bZIP transcription factor PERIANTHIA (PAN) plays a role in regulating stem cell fate by directly controlling AG expression and suggest that this activity is spatially restricted to the centermost region of the AG expression domain. These results suggest that the termination of floral stem cell fate is a multiply redundant process involving loci with unrelated floral patterning functions.

Mechanical stress contributes to the expression of the STM homeobox gene in Arabidopsis shoot meristems

The role of mechanical signals in cell identity determination remains poorly explored in tissues. Furthermore, because mechanical stress is widespread, mechanical signals are difficult to uncouple from biochemical-based transduction pathways. Here we focus on the homeobox gene SHOOT MERISTEMLESS (STM), a master regulator and marker of meristematic identity in Arabidopsis. We found that STM expression is quantitatively correlated to curvature in the saddle-shaped boundary domain of the shoot apical meristem. As tissue folding reflects the presence of mechanical stress, we test and demonstrate that STM expression is induced after micromechanical perturbations. We also show that STM expression in the boundary domain is required for organ separation. While STM expression correlates with auxin depletion in this domain, auxin distribution and STM expression can also be uncoupled. STM expression and boundary identity are thus strengthened through a synergy between auxin depletion and an auxin-independent mechanotransduction pathway at the shoot apical meristem. DOI: http://dx.doi.org/10.7554/eLife.07811.001