A novel protein Moat prevents ectopic epithelial folding by limiting Bazooka/Par3-dependent adherens junctions.

Contractile myosin and cell adhesion work together to induce tissue shape changes, but how they are patterned to achieve diverse morphogenetic outcomes remains unclear. Epithelial folding occurs via apical constriction, mediated by apical contractile myosin engaged with adherens junctions, as in Drosophila ventral furrow formation. While it has been shown that a multicellular gradient of myosin contractility determines folding shape, the impact of multicellular patterning of adherens junction levels on tissue folding is unknown. We identified a novel Drosophila gene moat essential for differential apical constriction and folding behaviors across the ventral epithelium which contains both folding ventral furrow and non-folding ectodermal anterior midgut (ectoAMG). We show that Moat functions to downregulate polarity-dependent adherens junctions through inhibiting cortical clustering of Bazooka/Par3 proteins. Such downregulation of polarity-dependent junctions is critical for establishing a myosin-dependent pattern of adherens junctions, which in turn mediates differential apical constriction in the ventral epithelium. In moat mutants, abnormally high levels of polarity-dependent junctions promote ectopic apical constriction in cells with low-level contractile myosin, resulting in expansion of infolding from ventral furrow to ectoAMG, and flattening of ventral furrow constriction gradient. Our results demonstrate that tissue-scale distribution of adhesion levels patterns apical constriction and establishes morphogenetic boundaries. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

A novel protein Moat prevents ectopic epithelial folding by limiting Bazooka/Par3-dependent adherens junctions.

Cortical myosin contraction and cell adhesion work together to promote tissue shape changes, but how they are modulated to achieve diverse morphogenetic outcomes remains unclear. Epithelial folding occurs via apical constriction, mediated by apical accumulation of contractile myosin engaged with adherens junctions, as in Drosophila ventral furrow formation. While levels of contractile myosin correlate with apical constriction, whether levels of adherens junctions modulate apical constriction is unknown. We identified a novel Drosophila gene moat that maintains low levels of Bazooka/Par3-dependent adherens junctions and thereby restricts apical constriction to ventral furrow cells with high-level contractile myosin. In moat mutants, abnormally high levels of Bazooka/Par3-dependent adherens junctions promote ectopic apical constriction in cells with low-level contractile myosin, insufficient for apical constriction in wild type. Such ectopic apical constriction expands infolding behavior from ventral furrow to ectodermal anterior midgut, which normally forms a later circular invagination. In moat mutant ventral furrow, a perturbed apical constriction gradient delays infolding. Our results indicate that levels of adherens junctions can modulate the outcome of apical constriction, providing an additional mechanism to define morphogenetic boundaries.

Digitally predicting protein localization and manipulating protein activity in fluorescence images using 4D reslicing GAN.

MOTIVATION: While multi-channel fluorescence microscopy is a vital imaging method in biological studies, the number of channels that can be imaged simultaneously is limited by technical and hardware limitations such as emission spectra cross-talk. One solution is using deep neural networks to model the localization relationship between two proteins so that the localization of one protein can be digitally predicted. Furthermore, the input and predicted localization implicitly reflect the modeled relationship. Accordingly, observing the response of the prediction via manipulating input localization could provide an informative way to analyze the modeled relationships between the input and the predicted proteins. RESULTS: We propose a protein localization prediction (PLP) method using a cGAN named 4D Reslicing Generative Adversarial Network (4DR-GAN) to digitally generate additional channels. 4DR-GAN models the joint probability distribution of input and output proteins by simultaneously incorporating the protein localization signals in four dimensions including space and time. Because protein localization often correlates with protein activation state, based on accurate PLP, we further propose two novel tools: digital activation (DA) and digital inactivation (DI) to digitally activate and inactivate a protein, in order to observing the response of the predicted protein localization. Compared with genetic approaches, these tools allow precise spatial and temporal control. A comprehensive experiment on six pairs of proteins shows that 4DR-GAN achieves higher-quality PLP than Pix2Pix, and the DA and DI responses are consistent with the known protein functions. The proposed PLP method helps simultaneously visualize additional proteins, and the developed DA and DI tools provide guidance to study localization-based protein functions. AVAILABILITY AND IMPLEMENTATION: The open-source code is available at https://github.com/YangJiaoUSA/4DR-GAN. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Live Imaging of Epithelial-Mesenchymal Transition in Mesoderm Cells of Gastrulating Drosophila Embryos.

Epithelial-mesenchymal transitions (EMTs) drive the generation of cell diversity during both evolution and development. More and more evidence has pointed to a model where EMT is not a binary switch but a reversible process that can be stabilized at intermediate states. Despite our vast knowledge on the signaling pathways that trigger EMT, we know very little about how EMT happens in a step-wise manner. Live imaging of cells that are undergoing EMT in intact, living, animals will provide us valuable insights into how EMT is executed at both the cellular and molecular levels and help us identify and understand the intermediate states. Here, we describe how to image early stages of EMT in the mesoderm cells of live Drosophila melanogaster embryos and how to image contractile myosin that suspends EMT progression.

Transcriptomic analysis of rice aleurone cells identified a novel abscisic acid response element.

Seeds serve as a great model to study plant responses to drought stress, which is largely mediated by abscisic acid (ABA). The ABA responsive element (ABRE) is a key cis-regulatory element in ABA signalling. However, its consensus sequence (ACGTG(G/T)C) is present in the promoters of only about 40% of ABA-induced genes in rice aleurone cells, suggesting other ABREs may exist. To identify novel ABREs, RNA sequencing was performed on aleurone cells of rice seeds treated with 20 µM ABA. Gibbs sampling was used to identify enriched elements, and particle bombardment-mediated transient expression studies were performed to verify the function. Gene ontology analysis was performed to predict the roles of genes containing the novel ABREs. This study revealed 2443 ABA-inducible genes and a novel ABRE, designated as ABREN, which was experimentally verified to mediate ABA signalling in rice aleurone cells. Many of the ABREN-containing genes are predicted to be involved in stress responses and transcription. Analysis of other species suggests that the ABREN may be monocot specific. This study also revealed interesting expression patterns of genes involved in ABA metabolism and signalling. Collectively, this study advanced our understanding of diverse cis-regulatory sequences and the transcriptomes underlying ABA responses in rice aleurone cells.

Three WRKY transcription factors additively repress abscisic acid and gibberellin signaling in aleurone cells.

Members of the WRKY transcription factor superfamily are essential for the regulation of many plant pathways. Functional redundancy due to duplications of WRKY transcription factors, however, complicates genetic analysis by allowing single-mutant plants to maintain wild-type phenotypes. Our analyses indicate that three group I WRKY genes, OsWRKY24, -53, and -70, act in a partially redundant manner. All three showed characteristics of typical WRKY transcription factors: each localized to nuclei and yeast one-hybrid assays indicated that they all bind to W-boxes, including those present in their own promoters. Quantitative real time-PCR (qRT-PCR) analyses indicated that the expression levels of the three WRKY genes varied in the different tissues tested. Particle bombardment-mediated transient expression analyses indicated that all three genes repress the GA and ABA signaling in a dosage-dependent manner. Combination of all three WRKY genes showed additive antagonism of ABA and GA signaling. These results suggest that these WRKY proteins function as negative transcriptional regulators of GA and ABA signaling. However, different combinations of these WRKY genes can lead to varied strengths in suppression of their targets.

RNA-sequencing reveals previously unannotated protein- and microRNA-coding genes expressed in aleurone cells of rice seeds.

The rice genome annotation has been greatly improved in recent years, largely due to the availability of full length cDNA sequences derived from many tissues. Among those yet to be studied is the aleurone layer, which produces hydrolases for mobilization of seed storage reserves during seed germination and post germination growth. Herein, we report transcriptomes of aleurone cells treated with the hormones abscisic acid, gibberellic acid, or both. Using a comprehensive approach, we identified hundreds of novel genes. To minimize the number of false positives, only transcripts that did not overlap with existing annotations, had a high level of expression, and showed a high level of uniqueness within the rice genome were considered to be novel genes. This approach led to the identification of 553 novel genes that encode proteins and/or microRNAs. The transcriptome data reported here will help to further improve the annotation of the rice genome.

Overexpression of maize mitogen-activated protein kinase gene, ZmSIMK1 in Arabidopsis increases tolerance to salt stress.

Mitogen-activated protein kinase (MAPK) cascades play a remarkably crucial role in plants. It has been studied intensively in model plants Arabidopsis, tobacco and rice. However, the function of MAPKs in maize (Zea mays L.) has not been well documented. ZmSIMK1 (Zea mays salt-induced mitogen-activated protein kinase 1) is a previously identified MAPK gene in maize. In this research, we charactered ZmSIMK1 and showed that ZmSIMK1 was involved in Arabidopsis salt stress. The genomic organization of ZmSIMK1 gene and its expression in maize have been analyzed. In order to investigate the function of ZmSIMK1, we generated transgenic Arabidopsis constitutively overexpressing ZmSIMK1. Ectopic expression of ZmSIMK1 in Arabidopsis resulted in increased resistance against salt stress. Importantly, ZmSIMK1-overexpressing Arabidopsis exhibited constitutive expression of stress-responsive marker genes, RD29A and P5CS1. Furthermore, RD29A and P5CS1 were upregulated under salt stress. These results suggest that ZmSIMK1 may play an important role in plant salt stress.

Abscisic acid and hydrogen peroxide induce a novel maize group C MAP kinase gene, ZmMPK7, which is responsible for the removal of reactive oxygen species.

Mitogen-activated protein kinase (MAPK) cascades are involved in biotic and abiotic stress responses. In plants, MAPKs are classified into four groups, designated A-D. Information about group C MAPKs is limited, and, in particular, no data from maize are available. In this article, we isolated a novel group C MAPK gene, ZmMPK7, from Zea mays. Exogenous abscisic acid (ABA) and hydrogen peroxide (H(2)O(2)) induced calcium-dependant transcription of ZmMPK7. Induction of this gene in response to ABA was blocked by several reactive oxygen species (ROS) manipulators such as imidazole, Tiron, and dimethylthiourea (DMTU). This result indicates that endogenous H(2)O(2) may be required for ZmMPK7-mediated ABA signaling. Expression of ZmMPK7 in Nicotonia tobaccum caused less H(2)O(2) to accumulate and alleviated ROS-mediated injuries following submission of the plants to osmotic stress. The enhanced total peroxidase (POD) activity in transgenic tobacco plants may contribute to removal of ROS. Finally, we have shown that the ZmMPK7 protein localizes in the nucleus. These results broaden our knowledge regarding plant group C MAPK activity in response to stress signals.

Expression of MaMAPK gene in seedlings of Malus L. under water stress.

Seedlings of three species of Malus were used to study the expression of mitogen-activated protein kinase (MAPK) in response to water stress: Malus hupehensis, a drought-sensitive species; Malus sieversii, a drought-tolerant species; and Malus micromalus, a middle type. Results showed that Malus MAPK (MaMAPK, GenBank accession No. AF435805) was expressed in both roots and leaves of seedlings of the three Malus species treated with 20% polyethylene glycol for different time periods. Expression levels peaked at 1.5 h after treatment with polyethylene glycol, then decreased to their lowest levels. Liquid kinase assays indicated that the dynamic changes of MAPK activity were very similar to those of the relative expression of MaMAPK mRNA. However, the peak of the former occurred slightly behind the latter. It was noticed that, although the kinase activity decreased after the peak, it was still higher than that of the control during the whole time period. These results suggested that MaMAPK was regulated not only by water stress at the transcription level, but also by phosphorylation and dephosphorylation at the protein level. In addition, of these three apple species, the highest MAPK activity and MaMAPK expression level was found in M. sieversii, followed by M. micromalus and M. hupehensis, suggesting that MAPK might be correlated with drought tolerance in these three species. The different expression levels might be one of the molecular mechanisms of the different drought tolerances in Malus.

Molecular cloning and expression of a gene encoding alcohol acyltransferase (MdAAT2) from apple (cv. Golden Delicious).

Volatile esters are major aroma components of apple, and an alcohol acyltransferase (AAT) catalyzes the final step in ester biosynthesis. The gene MdAAT2, which encodes a predicted 51.2 kDa protein containing features of other acyl transferases, was isolated from Malus domestica Borkh. (cv. Golden Delicious). In contrast to other apple varieties, the MdAAT2 gene of Golden Delicious is exclusively expressed in the fruit. The MdAAT2 protein is about 47.9 kDa and mainly localized in the fruit peel, as indicated by immunoblot and immunolocalization analysis. Northern blot and immunoblot analysis showed that the transcription and translation of MdAAT2 have a positive correlation with apple AAT enzyme activity and ester production, except in the later ripening stage, suggesting that MdAAT2 is involved in the regulation of ester biosysthesis and that a post-translation modification may be involved in regulation of AAT enzyme activity. Tissue disk assays of fruit peel revealed that using extraneous alcohols can recover the corresponding ester formation. Transcription and translation of MdAAT2 were both depressed by 1-methylcyclopropene (1-MCP) treatment and subsequent ester production was also prevented. These results suggest that: (1) ester production is mainly regulated by MdAAT2; (2) ethylene is also involved in this regulatory progress and (3) ester compounds rely principally on the availability of substrates.

[Cloning and expression characteristics of protein phosphatase gene ZmPP2C of Zea mays roots].

According to the conserved motifs of plant protein phosphatase 2C gene, degenerate oligonucleotides were designed. A full cDNA sequence of PP2C gene from Zea mays L. roots was cloned by RT-PCR. It was named ZmPP2C and had 936 bp. Southern blot showed that the ZmPP2C gene was a low copy in the Zea mays genome, and there was a small PP2C gene family. Northern blot showed that the expression of ZmPP2C gene was significantly different among Zea mays tissues. Zea mays roots treated with CaCl(2), MgCl(2), PEG, EGTA, and ABA for 24 h, the ZmPP2C expression increased only by CaCl(2) treatment. It showed the transcription of ZmPP2C gene was induced by Ca(2+), or in a Ca(2+)-dependent manner.