Two orthogonal differentiation gradients locally coordinate fruit morphogenesis.

Morphogenesis requires the coordination of cellular behaviors along developmental axes. In plants, gradients of growth and differentiation are typically established along a single longitudinal primordium axis to control global organ shape. Yet, it remains unclear how these gradients are locally adjusted to regulate the formation of complex organs that consist of diverse tissue types. Here we combine quantitative live imaging at cellular resolution with genetics, and chemical treatments to understand the formation of Arabidopsis thaliana female reproductive organ (gynoecium). We show that, contrary to other aerial organs, gynoecium shape is determined by two orthogonal, time-shifted differentiation gradients. An early mediolateral gradient controls valve morphogenesis while a late, longitudinal gradient regulates style differentiation. Local, tissue-dependent action of these gradients serves to fine-tune the common developmental program governing organ morphogenesis to ensure the specialized function of the gynoecium.

Live-imaging provides an atlas of cellular growth dynamics in the stamen.

Development of multicellular organisms is a complex process involving precise coordination of growth among individual cells. Understanding organogenesis requires measurements of cellular behaviors over space and time. In plants, such a quantitative approach has been successfully used to dissect organ development in both leaves and external floral organs, such as sepals. However, the observation of floral reproductive organs is hampered as they develop inside tightly closed floral buds, and are therefore difficult to access for imaging. We developed a confocal time-lapse imaging method, applied here to Arabidopsis (Arabidopsis thaliana), which allows full quantitative characterization of the development of stamens, the male reproductive organs. Our lineage tracing reveals the early specification of the filament and the anther. Formation of the anther lobes is associated with a temporal increase of growth at the lobe surface that correlates with intensive growth of the developing locule. Filament development is very dynamic and passes through three distinct phases: (1) initial intense, anisotropic growth, and high cell proliferation; (2) restriction of growth and proliferation to the filament proximal region; and (3) resumption of intense and anisotropic growth, displaced to the distal portion of the filament, without cell proliferation. This quantitative atlas of cellular growth dynamics provides a solid framework for future studies into stamen development.

The Relationship between AGAMOUS and Cytokinin Signaling in the Establishment of Carpeloid Features.

Gynoecium development is dependent on gene regulation and hormonal pathway interactions. The phytohormones auxin and cytokinin are involved in many developmental programs, where cytokinin is normally important for cell division and meristem activity, while auxin induces cell differentiation and organ initiation in the shoot. The MADS-box transcription factor AGAMOUS (AG) is important for the development of the reproductive structures of the flower. Here, we focus on the relationship between AG and cytokinin in Arabidopsis thaliana, and use the weak ag-12 and the strong ag-1 allele. We found that cytokinin induces carpeloid features in an AG-dependent manner and the expression of the transcription factors CRC, SHP2, and SPT that are involved in carpel development. AG is important for gynoecium development, and contributes to regulating, or else directly regulates CRC, SHP2, and SPT. All four genes respond to either reduced or induced cytokinin signaling and have the potential to be regulated by cytokinin via the type-B ARR proteins. We generated a model of a gene regulatory network, where cytokinin signaling is mainly upstream and in parallel with AG activity.

tasiR-ARFs Production and Target Regulation during In Vitro Maize Plant Regeneration.

During in vitro maize plant regeneration somatic cells change their normal fate and undergo restructuring to generate pluripotent cells able to originate new plants. Auxins are essential to achieve such plasticity. Their physiological effects are mediated by auxin response factors (ARFs) that bind auxin responsive elements within gene promoters. Small trans-acting (ta)-siRNAs, originated from miR390-guided TAS3 primary transcript cleavage, target ARF3/4 class (tasiR-ARFs). Here we found that TAS3b precursor as well as derived tasiR-ARFbD5 and tasiR-ARFbD6 display significantly lower levels in non-embryogenic callus (NEC), while TAS3g, miR390 and tasiR-ARFg are more abundant in the same tissue. However, Argonaute (AGO7) and leafbladeless 1 (LBLl) required for tasiR-ARF biogenesis showed significantly higher transcript levels in EC suggesting limited tasiR-ARF biogenesis in NEC. The five maize ARFs targeted by tasiR-ARFs were also significantly enriched in EC and accompanied by higher auxin accumulation with punctuate patterns in this tissue. At hormone half-reduction and photoperiod implementation, plant regeneration initiated from EC with transient TAS3g, miR390 and tasiR-ARFg increase. Upon complete hormone depletion, TAS3b became abundant and derived tasiR-ARFs gradually increased at further regeneration stages. ZmARF transcripts targeted by tasiR-ARFs, as well as AGO7 and LBL1 showed significantly lower levels during regeneration than in EC. These results indicate a dynamic tasiR-ARF mediated regulation throughout maize in vitro plant regeneration.

Gynoecium development: networks in Arabidopsis and beyond.

Life has always found a way to preserve itself. One strategy that has been developed for this purpose is sexual reproduction. In land plants, the gynoecium is considered to be at the top of evolutionary innovation, since it has been a key factor in the success of the angiosperms. The gynoecium is composed of carpels with different tissues that need to develop and differentiate in the correct way. In order to control and guide gynoecium development, plants have adapted elements of pre-existing gene regulatory networks (GRNs) but new ones have also evolved. The GRNs can interact with internal factors (e.g. hormones and other metabolites) and external factors (e.g. mechanical signals and temperature) at different levels, giving robustness and flexibility to gynoecium development. Here, we review recent findings regarding the role of cytokinin-auxin crosstalk and the genes that connect these hormonal pathways during early gynoecium development. We also discuss some examples of internal and external factors that can modify GRNs. Finally, we make a journey through the flowering plant lineage to determine how conserved are these GRNs that regulate gynoecium and fruit development.

A Simple Protocol for Imaging Floral Tissues of Arabidopsis with Confocal Microscopy.

We present a simple protocol to image floral tissues with confocal laser scanning microscopy (CLSM). Recently, new imaging techniques have emerged that improve the image quality of plant tissues. In this protocol, as an example, we focus on the fluorescence detection of the miRNA MIR164c precursor. Briefly, the method involves tissue clearing, cell wall staining, and the visualization of fluorescence in tissues in young floral buds of Arabidopsis with CLSM with the use of water dipping lenses.

New roles of NO TRANSMITTING TRACT and SEEDSTICK during medial domain development in Arabidopsis fruits.

The gynoecium, the female reproductive part of the flower, is key for plant sexual reproduction. During its development, inner tissues such as the septum and the transmitting tract tissue, important for pollen germination and guidance, are formed. In Arabidopsis, several transcription factors are known to be involved in the development of these tissues. One of them is NO TRANSMITTING TRACT (NTT), essential for transmitting tract formation. We found that the NTT protein can interact with several gynoecium-related transcription factors, including several MADS-box proteins, such as SEEDSTICK (STK), known to specify ovule identity. Evidence suggests that NTT and STK control enzyme and transporter-encoding genes involved in cell wall polysaccharide and lipid distribution in gynoecial medial domain cells. The results indicate that the simultaneous loss of NTT and STK activity affects polysaccharide and lipid deposition and septum fusion, and delays entry of septum cells to their normal degradation program. Furthermore, we identified KAWAK, a direct target of NTT and STK, which is required for the correct formation of fruits in Arabidopsis These findings position NTT and STK as important factors in determining reproductive competence.

Non-destructive Plant Morphometric and Color Analyses Using an Optoelectronic 3D Color Microscope.

Gene function discovery in plants, as other plant science quests, is aided by tools that image, document, and measure plant phenotypes. Tools that acquire images of plant organs and tissues at the microscopic level have evolved from qualitative documentation tools, to advanced tools where software-assisted analysis of images extracts quantitative information that allows statistical analyses. They are useful to perform morphometric studies that describe plant physical characteristics and quantify phenotypes, aiding gene function discovery. In parallel, non-destructive, versatile, robust, and user friendly technologies have also been developed for surface topography analysis and quality control in the industrial manufacture sector, such as optoelectronic three-dimensional (3D) color microscopes. These microscopes combine optical lenses, electronic image sensors, motorized stages, graphics engines, and user friendly software to allow the visualization and inspection of objects of diverse sizes and shapes from different angles. This allow the integration of different automatically obtained images along the Z axis of an object, into a single image with a large depth-of-field, or a 3D model in color. In this work, we explored the performance of an optoelectronic microscope to study plant morphological phenotypes and plant surfaces in different model species. Furthermore, as a "proof-of-concept," we included the phenotypic characterization (morphometric analyses at the organ level, color, and cell size measurements) of Arabidopsis mutant leaves. We found that the microscope tested is a suitable, practical, and fast tool to routinely and precisely analyze different plant organs and tissues, producing both high-quality, sharp color images and morphometric and color data in real time. It is fully compatible with live plant tissues (no sample preparation is required) and does not require special conditions, high maintenance, nor complex training. Therefore, though barely reported in plant scientific studies, optoelectronic microscopes should emerge as convenient and useful tools for phenotypic characterization in plant sciences.