Molecular Regulation of the Annual Growth Cycle in Populus Trees

Adaptation to the change of seasons is essential for tree survival. Here I show that the phenology of hybrid aspen is regulated by three FLOWERING LOCUS T (FT) genes. FT1, FT2a and FT2b are the result of both a whole genome and a local duplication. All three FTs are highly similar in sequence but their expression patterns and functions have diverged over time. FT1 expression is drastically induced by cold temperatures during winter in vegetative and reproductive buds, while FT2a and FT2b are expressed in leaves during spring and summer. I used CRISPR/Cas9 gene editing tools to generate individual and specific knockout mutants of FT1 and FT2. FT1 mutants showed no defects in vegetative growth during the first year. However, their bud flush was severely delayed, indicating a role of FT1 in dormancy release during winter. In contrast, knock-out of both FT2s greatly impaired growth and lead to early growth cessation, showing their importance for vegetative growth during summer. Additionally, I investigated the regulation of FT and possible mechanisms that can fine-tune the response to seasonal changes. I show that the timing of both bud set and bud flush is regulated by the photoreceptor Phytochrome B and its interacting factor PHYTOCHROME INTERACTING FACTOR 8 trough FT2 and probably also FT1. Furthermore, I show that growth cessation is induced in response to SD by SHORT VEGETATIVE PHASE LIKE, which represses the expression of FT2 and gibberellin metabolism genes in the leaves

Populus SVL Acts in Leaves to Modulate the Timing of Growth Cessation and Bud Set

SHORT VEGETATIVE PHASE (SVP) is an important regulator of FLOWERING LOCUS T (FT) in the thermosensory pathway of Arabidopsis. It is a negative regulator of flowering and represses FT transcription. In poplar trees, FT2 is central for the photoperiodic control of growth cessation, which also requires the decrease of bioactive gibberellins (GAs). In angiosperm trees, genes similar to SVP, sometimes named DORMANCY-ASSOCIATED MADS-BOX genes, control temperature-mediated bud dormancy. Here we show that SVL, an SVP ortholog in aspen trees, besides its role in controlling dormancy through its expression in buds, is also contributing to the regulation of short day induced growth cessation and bud set through its expression in leaves. SVL is upregulated during short days in leaves and binds to the FT2 promoter to repress its transcription. It furthermore decreases the amount of active GAs, whose downregulation is essential for growth cessation, by repressing the transcription of GA20 oxidase. Finally, the SVL protein is more stable in colder temperatures, thus integrating the temperature signal into the response. We conclude that the molecular function of SVL in the photoperiodic pathway has been conserved between Arabidopsis and poplar trees, albeit the physiological process it controls has changed. SVL is thus both involved in regulating the photoperiod response in leaves, modulating the timing of growth cessation and bud set, and in the subsequent temperature regulation of dormancy in the buds

FLOWERING LOCUS T paralogs control the annual growth cycle in Populus trees

In temperate and boreal regions, perennials adapt their annual growth cycle to the change of seasons. These adaptations ensure survival in harsh environmental conditions, allowing growth at different latitudes and altitudes, and are therefore tightly regulated. Populus tree species cease growth and form terminal buds in autumn when photoperiod falls below a certain threshold.(1) This is followed by establishment of dormancy and cold hardiness over the winter. At the center of the photoperiodic pathway in Populus is the gene FLOWERING LOCUS T2 (FT2), which is expressed during summer and harbors significant SNPs in its locus associated with timing of bud set.(1-4) The paralogous gene FT1, on the other hand, is hyper-induced in chilling buds during winter.(3,5) Even though its function is so far unknown, it has been suggested to be involved in the regulation of flowering and the release of winter dormancy(.3,5). In this study, we employ CRISPR-Cas9-mediated gene editing to individually study the function of the FT-like genes in Populus trees. We show that while FT2 is required for vegetative growth during spring and summer and regulates the entry into dormancy, expression of FT1 is absolutely required for bud flush in spring. Gene expression profiling suggests that this function of FT1 is linked to the release of winter dormancy rather than to the regulation of bud flush per se. These data show how FT duplication and sub-functionalization have allowed Populus trees to regulate two completely different and major developmental control points during the yearly growth cycle

Preoperative Brain Tumor Imaging: Models and Software for Segmentation and Standardized Reporting

For patients suffering from brain tumor, prognosis estimation and treatment decisions are made by a multidisciplinary team based on a set of preoperative MR scans. Currently, the lack of standardized and automatic methods for tumor detection and generation of clinical reports, incorporating a wide range of tumor characteristics, represents a major hurdle. In this study, we investigate the most occurring brain tumor types: glioblastomas, lower grade gliomas, meningiomas, and metastases, through four cohorts of up to 4,000 patients. Tumor segmentation models were trained using the AGU-Net architecture with different preprocessing steps and protocols. Segmentation performances were assessed in-depth using a wide-range of voxel and patient-wise metrics covering volume, distance, and probabilistic aspects. Finally, two software solutions have been developed, enabling an easy use of the trained models and standardized generation of clinical reports: Raidionics and Raidionics-Slicer. Segmentation performances were quite homogeneous across the four different brain tumor types, with an average true positive Dice ranging between 80 and 90%, patient-wise recall between 88 and 98%, and patient-wise precision around 95%. In conjunction to Dice, the identified most relevant other metrics were the relative absolute volume difference, the variation of information, and the Hausdorff, Mahalanobis, and object average symmetric surface distances. With our Raidionics software, running on a desktop computer with CPU support, tumor segmentation can be performed in 16–54 s depending on the dimensions of the MRI volume. For the generation of a standardized clinical report, including the tumor segmentation and features computation, 5–15 min are necessary. All trained models have been made open-access together with the source code for both software solutions and validation metrics computation. In the future, a method to convert results from a set of metrics into a final single score would be highly desirable for easier ranking across trained models. In addition, an automatic classification of the brain tumor type would be necessary to replace manual user input. Finally, the inclusion of post-operative segmentation in both software solutions will be key for generating complete post-operative standardized clinical reports.publishedVersio

Segmentation of glioblastomas in early post-operative multi-modal MRI with deep neural networks

Extent of resection after surgery is one of the main prognostic factors for patients diagnosed with glioblastoma. To achieve this, accurate segmentation and classification of residual tumor from post-operative MR images is essential. The current standard method for estimating it is subject to high inter- and intra-rater variability, and an automated method for segmentation of residual tumor in early post-operative MRI could lead to a more accurate estimation of extent of resection. In this study, two state-of-the-art neural network architectures for pre-operative segmentation were trained for the task. The models were extensively validated on a multicenter dataset with nearly 1000 patients, from 12 hospitals in Europe and the United States. The best performance achieved was a 61\% Dice score, and the best classification performance was about 80\% balanced accuracy, with a demonstrated ability to generalize across hospitals. In addition, the segmentation performance of the best models was on par with human expert raters. The predicted segmentations can be used to accurately classify the patients into those with residual tumor, and those with gross total resection.Comment: 13 pages, 4 figures, 4 table

Preoperative Brain Tumor Imaging: Models and Software for Segmentation and Standardized Reporting

For patients suffering from brain tumor, prognosis estimation and treatment decisions are made by a multidisciplinary team based on a set of preoperative MR scans. Currently, the lack of standardized and automatic methods for tumor detection and generation of clinical reports, incorporating a wide range of tumor characteristics, represents a major hurdle. In this study, we investigate the most occurring brain tumor types: glioblastomas, lower grade gliomas, meningiomas, and metastases, through four cohorts of up to 4,000 patients. Tumor segmentation models were trained using the AGU-Net architecture with different preprocessing steps and protocols. Segmentation performances were assessed in-depth using a wide-range of voxel and patient-wise metrics covering volume, distance, and probabilistic aspects. Finally, two software solutions have been developed, enabling an easy use of the trained models and standardized generation of clinical reports: Raidionics and Raidionics-Slicer. Segmentation performances were quite homogeneous across the four different brain tumor types, with an average true positive Dice ranging between 80 and 90%, patient-wise recall between 88 and 98%, and patient-wise precision around 95%. In conjunction to Dice, the identified most relevant other metrics were the relative absolute volume difference, the variation of information, and the Hausdorff, Mahalanobis, and object average symmetric surface distances. With our Raidionics software, running on a desktop computer with CPU support, tumor segmentation can be performed in 16–54 s depending on the dimensions of the MRI volume. For the generation of a standardized clinical report, including the tumor segmentation and features computation, 5–15 min are necessary. All trained models have been made open-access together with the source code for both software solutions and validation metrics computation. In the future, a method to convert results from a set of metrics into a final single score would be highly desirable for easier ranking across trained models. In addition, an automatic classification of the brain tumor type would be necessary to replace manual user input. Finally, the inclusion of post-operative segmentation in both software solutions will be key for generating complete post-operative standardized clinical reports

Phytochrome B and PHYTOCHROME INTERACTING FACTOR8 modulate seasonal growth in trees

The seasonally synchronized annual growth cycle that is regulated mainly by photoperiod and temperature cues is a crucial adaptive strategy for perennial plants in boreal and temperate ecosystems.Phytochrome B (phyB), as a light and thermal sensor, has been extensively studied in Arabidopsis. However, the specific mechanisms for how the phytochrome photoreceptors control the phenology in tree species remain poorly understood.We characterized the functions of PHYB genes and their downstream PHYTOCHROME INTERACTING FACTOR (PIF) targets in the regulation of shade avoidance and seasonal growth in hybrid aspen trees. We show that while phyB1 and phyB2, as phyB in other plants, act as suppressors of shoot elongation during vegetative growth, they act as promoters of tree seasonal growth. Furthermore, while the Populus homologs of both PIF4 and PIF8 are involved in the shade avoidance syndrome (SAS), only PIF8 plays a major role as a suppressor of seasonal growth.Our data suggest that the PHYB-PIF8 regulon controls seasonal growth through the regulation of FT and CENL1 expression while a genome-wide transcriptome analysis suggests how, in Populus trees, phyB coordinately regulates SAS responses and seasonal growth cessation

GIGANTEA influences leaf senescence in trees in two different ways

GIGANTEA (GI) genes have a central role in plant development and influence several processes. Hybrid aspen T89 (Populus tremula x tremuloides) trees with low GI expression engineered through RNAi show severely compromised growth. To study the effect of reduced GI expression on leaf traits with special emphasis on leaf senescence, we grafted GI-RNAi scions onto wild-type rootstocks and successfully restored growth of the scions. The RNAi line had a distorted leaf shape and reduced photosynthesis, probably caused by modulation of phloem or stomatal function, increased starch accumulation, a higher carbon-to-nitrogen ratio, and reduced capacity to withstand moderate light stress. GI-RNAi also induced senescence under long day (LD) and moderate light conditions. Furthermore, the GI-RNAi lines were affected in their capacity to respond to "autumn environmental cues" inducing senescence, a type of leaf senescence that has physiological and biochemical characteristics that differ from those of senescence induced directly by stress under LD conditions. Overexpression of GI delayed senescence under simulated autumn conditions. The two different effects on leaf senescence under LD or simulated autumn conditions were not affected by the expression of FLOWERING LOCUS T. GI expression regulated leaf senescence locally-the phenotype followed the genotype of the branch, independent of its position on the tree-and trees with modified gene expression were affected in a similar way when grown in the field as under controlled conditions. Taken together, GI plays a central role in sensing environmental changes during autumn and determining the appropriate timing for leaf senescence in Populus

METACASPASE9 modulates autophagy to confine cell death tothe target cells during Arabidopsis vascular xylem differentiation

We uncovered that the level of autophagy in plant cells undergoingprogrammed cell death determines the fate of the surrounding cells.Our approach consisted of using Arabidopsis thaliana cell culturescapable of differentiating into two different cell types: vasculartracheary elements (TEs) that undergo programmed cell death(PCD) and protoplast autolysis, and parenchymatic non-TEs thatremain alive. The TE cell type displayed higher levels of autophagywhen expression of the TE-specific METACASPASE9 (MC9) wasreduced using RNAi (MC9-RNAi). Misregulation of autophagy in theMC9-RNAi TEs coincided with ectopic death of the non-TEs, implyingthe existence of an autophagy-dependent intercellular signallingfrom within the TEs towards the non-TEs. Viability of the non-TEswas restored when AUTOPHAGY2 (ATG2) was downregulatedspecifically in MC9-RNAi TEs, demonstrating the importance ofautophagy in the spatial confinement of cell death. Our resultssuggest that other eukaryotic cells undergoing PCD might also needto tightly regulate their level of autophagy to avoid detrimentalconsequences for the surrounding cells.Bio4Energ

FLOWERING LOCUS T paralogs control the annual growth cycle in Populus trees

In temperate and boreal regions, perennials adapt their annual growth cycle to the change of seasons. These adaptations ensure survival in harsh environmental conditions, allowing growth at different latitudes and altitudes, and are therefore tightly regulated. Populus tree species cease growth and form terminal buds in autumn when photoperiod falls below a certain threshold.1 This is followed by establishment of dormancy and cold hardiness over the winter. At the center of the photoperiodic pathway in Populus is the gene FLOWERING LOCUS T2 (FT2), which is expressed during summer and harbors significant SNPs in its locus associated with timing of bud set.1–4 The paralogous gene FT1, on the other hand, is hyper-induced in chilling buds during winter.3,5 Even though its function is so far unknown, it has been suggested to be involved in the regulation of flowering and the release of winter dormancy.3,5 In this study, we employ CRISPR-Cas9-mediated gene editing to individually study the function of the FT-like genes in Populus trees. We show that while FT2 is required for vegetative growth during spring and summer and regulates the entry into dormancy, expression of FT1 is absolutely required for bud flush in spring. Gene expression profiling suggests that this function of FT1 is linked to the release of winter dormancy rather than to the regulation of bud flush per se. These data show how FT duplication and sub-functionalization have allowed Populus trees to regulate two completely different and major developmental control points during the yearly growth cycle