HIGHLIGHTThe chromatin-modifying complex Paf1 is required for shoot and root thigmomorphogenesis and touch-inducible gene expression.

Although accumulating evidence suggests that gene regulation is highly stochastic, genetic screens have successfully uncovered master developmental regulators, questioning the relationship between transcriptional noise and intrinsic robustness of development. To identify developmental modules that are more or less resilient to large-scale genetic perturbations, we used the Arabidopsis polymerase II-associated factor 1 complex (Paf1c) mutant vip3, which is impaired in several RNA polymerase II-dependent transcriptional processes. We found that the control of flower termination was not as robust as classically pictured. In angiosperms, the floral female organs, called carpels, display determinate growth: their development requires the arrest of stem cell maintenance. In vip3 mutant flowers, carpels displayed a highly variable morphology, with different degrees of indeterminacy defects up to wild-type size inflorescence emerging from carpels. This phenotype was associated with variable expression of two key regulators of flower termination and stem cell maintenance in flowers, WUSCHEL and AGAMOUS. The phenotype was also dependent on growth conditions. Together, these results highlight the surprisingly plastic nature of stem cell maintenance in plants and its dependence on Paf1c.

In plants, aerial organs are initiated at stereotyped intervals, both spatially (every 137° in a pattern called phyllotaxis) and temporally (at prescribed time intervals called plastochrons). To investigate the molecular basis of such regularity, mutants with altered architecture have been isolated. However, most of them only exhibit plastochron defects and/or produce a new, albeit equally reproducible, phyllotactic pattern. This leaves open the question of a molecular control of phyllotaxis regularity. Here, we show that phyllotaxis regularity depends on the function of VIP proteins, components of the RNA polymerase II-associated factor 1 complex (Paf1c). Divergence angles between successive organs along the stem exhibited increased variance in vip3-1 and vip3-2 compared with the wild type, in two different growth conditions. Similar results were obtained with the weak vip3-6 allele and in vip6, a mutant for another Paf1c subunit. Mathematical analysis confirmed that these defects could not be explained solely by plastochron defects. Instead, increased variance in phyllotaxis in vip3 was observed at the meristem and related to defects in spatial patterns of auxin activity. Thus, the regularity of spatial, auxin-dependent, patterning at the meristem requires Paf1c.

The shoot apical meristem is the central organizer of plant aerial organogenesis. The molecular bases of its functions involve several cross-talks between transcription factors, hormones and microRNAs. We recently showed that the expression of the homeobox transcription factor STM is induced by mechanical perturbations, adding another layer of complexity to this regulation. Here we provide additional evidence that mechanical perturbations impact the promoter activity of CUC3, an important regulator of boundary formation at the shoot meristem. Interestingly, we did not detect such an effect for CUC1. This suggests that the robustness of expression patterns and developmental programs is controlled via a combined action of molecular factors as well as mechanical cues in the shoot apical meristem. KEYWORDSCUC genes; mechanical stress; Meristem; micro-RNAs Throughout their lifetime, plants are exposed to a number of external mechanical perturbations, such as wind or touch. This leads to changes in mRNA level of many genes, and long-term developmental responses such as stem thickening and flowering delay 5 In addition to these external factors, plants are also constantly affected by intrinsic tensile stresses, notably because plant cells are under high turgor pressure. There is now accumulating evidence that these internal stresses are affecting many aspects of the cells, thus channeling growth in the long term.14 The shoot apical meristem (SAM) is the central organizer of plant aerial organogenesis. Initiation of new organs and maintenance of SAM is achieved through the concomitant action of multiple regulatory pathways. Genetic screens have identified many key players, including transcription factors, hormones, and microRNAs. 38,48,53,55 In addition to these biochemical factors, mechanical forces are also present within the meristem. In particular, the boundary domain that separates newly emerging organ from the meristem is under highly anisotropic tensile stresses. 4,15,32 A key question for the future is the analysis of the interplay between mechanical forces and the molecular regulators of meristem function (Fig. 1A).In a recent article, 26 we showed that a master regulator of meristem maintenance, the homeodomain protein SHOOT MERISTEMLESS (STM) is expressed at a higher level in the boundary domain, and that this local increase in promoter activity can be related to mechanical stress: mechanical perturbations are sufficient to induce STM expression in the meristem. Interestingly, mechanical perturbations do not affect all boundary-expressed genes in the same way. For instance, the promoter activity of the PINOID gene, which is also increased in the boundary domain, is not affected by mechanical perturbations. 26Here we focus on the most canonical genetic markers of boundary identity, the CUP SHAPED COTYLEDON (CUC) genes. CUC1, CUC2 and CUC3, belong to a group of NAC domain transcription factors and show a high level of functional redundancy. They play an essential role in shoot meristem initiation throug...

To investigate the role of chromatin regulators in patterning gene expression, we employed a unique epigenetically controlled and highly tissue-specific green fluorescent protein reporter line in Arabidopsis (Arabidopsis thaliana). Using a combination of forward and reverse genetic approaches on this line, we show here that distinct epigenetic regulators are involved in silencing the transgene in different tissues. The forward genetic screen led to the identification of a novel HISTONE DEACETYLASE6 (HDA6) mutant allele (epigenetic control1, hda6-8). This allele differs from the previously reported alleles, as it did not affect DNA methylation and only had a very modest effect on the release of transposable elements and other heterochromatic transcripts. Overall, our data shows that HDA6 has at least two clearly separable activities in different genomic regions. In addition, we present an unexpected role for HDA6 in the control of DNA methylation at CG dinucleotides.

Artificial mechanical perturbations affect chromatin in animal cells in culture. Whether this is also relevant to growing tissues in living organisms remains debated. In plants, aerial organ emergence occurs through localized outgrowth at the periphery of the shoot apical meristem, which also contains a stem cell niche. Interestingly, organ outgrowth has been proposed to generate compression in the saddle-shaped organ–meristem boundary domain. Yet whether such growth-induced mechanical stress affects chromatin in plant tissues is unknown. Here, by imaging the nuclear envelope in vivo over time and quantifying nucleus deformation, we demonstrate the presence of active nuclear compression in that domain. We developed a quantitative pipeline amenable to identifying a subset of very deformed nuclei deep in the boundary and in which nuclei become gradually narrower and more elongated as the cell contracts transversely. In this domain, we find that the number of chromocenters is reduced, as shown by chromatin staining and labeling, and that the expression of linker histone H1.3 is induced. As further evidence of the role of forces on chromatin changes, artificial compression with a MicroVice could induce the ectopic expression of H1.3 in the rest of the meristem. Furthermore, while the methylation status of chromatin was correlated with nucleus deformation at the meristem boundary, such correlation was lost in the h1.3 mutant. Altogether, we reveal that organogenesis in plants generates compression that is able to have global effects on chromatin in individual cells.

In animals, it is now well established that forces applied at the cell surface are propagated through the cytoskeleton to the nucleus, leading to deformations of the nuclear structure and, potentially, to modification of gene expression. Consistently, altered nuclear mechanics has been related to many genetic disorders, such as muscular dystrophy, cardiomyopathy and progeria. In plants, the integration of mechanical signals in cell and developmental biology has also made great progress. Yet, while the link between cell wall stresses and cytoskeleton is consolidated, such cortical mechanical cues have not been integrated with the nucleoskeleton. Here, we propose to take inspiration from studies on animal nuclei to identify relevant methods amenable to probing nucleus mechanics and deformation in plant cells, with a focus on microrheology. To identify potential molecular targets, we also compare the players at the nuclear envelope, namely lamina and LINC complex, in both plant and animal nuclei. Understanding how mechanical signals are transduced to the nucleus across kingdoms will likely have essential implications in development (e.g. how mechanical cues add robustness to gene expression patterns), in the nucleoskeleton–cytoskeleton nexus (e.g. how stress is propagated in turgid/walled cells), as well as in transcriptional control, chromatin biology and epigenetics.

In present paper series of trimethine cyanines modified in 5,5'- or 6,6'- position with hydroxy- or methoxy- substituents is studied for their ability to interact selectively with fibrillar formations. Processes of dye aggregation that accompany this interaction were also investigated. Meso-methyl trimethynecyanines with 5,5'- methoxy (7519) and hydroxy (7515) substituents strongly (up to 40 times) increase fluorescence intensity in the presence of fibrillar insulin, and also give noticeable fluorescent response on the presence of various aggregated proteins (lysozyme, β-lactoglobulin, α-synuclein A53T). 7519 and 7515 dyes can be used for fluorometric detection of fibrillar insulin at concentrations of approximately 1.5-120 microg/ml. For meso-ethyl substituted dye 7514 the ability to form H- and J-aggregates upon interaction with insulin fibrils was suggested. The model of the H- and J-aggregate packing in the protein fibrillar structure has been proposed.