Endosperm cell death promoted by NAC transcription factors facilitates embryo invasion in Arabidopsis.

In flowering plants, two fertilization products develop within the limited space of the seed: the embryo and the surrounding nutritive endosperm. The final size of the endosperm is modulated by the degree of embryo growth. In Arabidopsis thaliana, the endosperm expands rapidly after fertilization, but later gets invaded by the embryo that occupies most of the seed volume at maturity, surrounded by a single remaining aleurone-like endosperm layer.1,2,3,4 Embryo invasion is facilitated by the endosperm-expressed bHLH-type transcription factor ZHOUPI, which promotes weakening of endosperm cell walls.5,6 Endosperm elimination in zou mutants is delayed, and embryo growth is severely affected; the endosperm finally collapses around the dwarf embryo, causing the shriveled appearance of mature zou seeds.5,6,7 However, whether ZHOUPI facilitates mechanical endosperm destruction by the invading embryo or whether an active programmed cell death (PCD) process causes endosperm elimination has been subject to debate.2,8 Here we show that developmental PCD controlled by multiple NAC transcription factors in the embryo-adjacent endosperm promotes gradual endosperm elimination. Misexpressing the NAC transcription factor KIRA1 in the entire endosperm caused total endosperm elimination, generating aleurone-less mature seeds. Conversely, dominant and recessive higher-order NAC mutants led to delayed endosperm elimination and impaired cell corpse clearance. Promoting PCD in the zhoupi mutant partially rescued its embryo growth defects, while the endosperm in a zhoupi nac higher-order mutant persisted until seed desiccation. These data suggest that a combination of cell wall weakening and PCD jointly facilitates embryo invasion by an active auto-elimination of endosperm cells.

Predicting yield of individual field-grown rapeseed plants from rosette-stage leaf gene expression.

In the plant sciences, results of laboratory studies often do not translate well to the field. To help close this lab-field gap, we developed a strategy for studying the wiring of plant traits directly in the field, based on molecular profiling and phenotyping of individual plants. Here, we use this single-plant omics strategy on winter-type Brassica napus (rapeseed). We investigate to what extent early and late phenotypes of field-grown rapeseed plants can be predicted from their autumnal leaf gene expression, and find that autumnal leaf gene expression not only has substantial predictive power for autumnal leaf phenotypes but also for final yield phenotypes in spring. Many of the top predictor genes are linked to developmental processes known to occur in autumn in winter-type B. napus accessions, such as the juvenile-to-adult and vegetative-to-reproductive phase transitions, indicating that the yield potential of winter-type B. napus is influenced by autumnal development. Our results show that single-plant omics can be used to identify genes and processes influencing crop yield in the field.

Spatial and temporal regulation of parent-of-origin allelic expression in the endosperm.

Genomic imprinting promotes differential expression of parental alleles in the endosperm of flowering plants and is regulated by epigenetic modification such as DNA methylation and histone tail modifications in chromatin. After fertilization, the endosperm develops through a syncytial stage before it cellularizes and becomes a nutrient source for the growing embryo. Regional compartmentalization has been shown both in early and late endosperm development, and different transcriptional domains suggest divergent spatial and temporal regional functions. The analysis of the role of parent-of-origin allelic expression in the endosperm as a whole and the investigation of domain-specific functions have been hampered by the inaccessibility of the tissue for high-throughput transcriptome analyses and contamination from surrounding tissue. Here, we used fluorescence-activated nuclear sorting (FANS) of nuclear targeted GFP fluorescent genetic markers to capture parental-specific allelic expression from different developmental stages and specific endosperm domains. This approach allowed us to successfully identify differential genomic imprinting with temporal and spatial resolution. We used a systematic approach to report temporal regulation of imprinted genes in the endosperm, as well as region-specific imprinting in endosperm domains. Analysis of our data identified loci that are spatially differentially imprinted in one domain of the endosperm, while biparentally expressed in other domains. These findings suggest that the regulation of genomic imprinting is dynamic and challenge the canonical mechanisms for genomic imprinting.

BREEDIT: a multiplex genome editing strategy to improve complex quantitative traits in maize.

Ensuring food security for an ever-growing global population while adapting to climate change is the main challenge for agriculture in the 21st century. Although new technologies are being applied to tackle this problem, we are approaching a plateau in crop improvement using conventional breeding. Recent advances in CRISPR/Cas9-mediated gene engineering have paved the way to accelerate plant breeding to meet this increasing demand. However, many traits are governed by multiple small-effect genes operating in complex interactive networks. Here, we present the gene discovery pipeline BREEDIT, which combines multiplex genome editing of whole gene families with crossing schemes to improve complex traits such as yield and drought tolerance. We induced gene knockouts in 48 growth-related genes into maize (Zea mays) using CRISPR/Cas9 and generated a collection of over 1,000 gene-edited plants. The edited populations displayed (on average) 5%-10% increases in leaf length and up to 20% increases in leaf width compared with the controls. For each gene family, edits in subsets of genes could be associated with enhanced traits, allowing us to reduce the gene space to be considered for trait improvement. BREEDIT could be rapidly applied to generate a diverse collection of mutants to identify promising gene modifications for later use in breeding programs.

Identification of growth regulators using cross-species network analysis in plants.

With the need to increase plant productivity, one of the challenges plant scientists are facing is to identify genes that play a role in beneficial plant traits. Moreover, even when such genes are found, it is generally not trivial to transfer this knowledge about gene function across species to identify functional orthologs. Here, we focused on the leaf to study plant growth. First, we built leaf growth transcriptional networks in Arabidopsis (Arabidopsis thaliana), maize (Zea mays), and aspen (Populus tremula). Next, known growth regulators, here defined as genes that when mutated or ectopically expressed alter plant growth, together with cross-species conserved networks, were used as guides to predict novel Arabidopsis growth regulators. Using an in-depth literature screening, 34 out of 100 top predicted growth regulators were confirmed to affect leaf phenotype when mutated or overexpressed and thus represent novel potential growth regulators. Globally, these growth regulators were involved in cell cycle, plant defense responses, gibberellin, auxin, and brassinosteroid signaling. Phenotypic characterization of loss-of-function lines confirmed two predicted growth regulators to be involved in leaf growth (NPF6.4 and LATE MERISTEM IDENTITY2). In conclusion, the presented network approach offers an integrative cross-species strategy to identify genes involved in plant growth and development.

An in situ sequencing approach maps PLASTOCHRON1 at the boundary between indeterminate and determinate cells.

The plant shoot apex houses the shoot apical meristem, a highly organized and active stem-cell tissue where molecular signaling in discrete cells determines when and where leaves are initiated. We optimized a spatial transcriptomics approach, in situ sequencing (ISS), to colocalize the transcripts of 90 genes simultaneously on the same section of tissue from the maize (Zea mays) shoot apex. The RNA ISS technology reported expression profiles that were highly comparable with those obtained by in situ hybridizations (ISHs) and allowed the discrimination between tissue domains. Furthermore, the application of spatial transcriptomics to the shoot apex, which inherently comprised phytomers that are in gradual developmental stages, provided a spatiotemporal sequence of transcriptional events. We illustrate the power of the technology through PLASTOCHRON1 (PLA1), which was specifically expressed at the boundary between indeterminate and determinate cells and partially overlapped with ROUGH SHEATH1 and OUTER CELL LAYER4 transcripts. Also, in the inflorescence, PLA1 transcripts localized in cells subtending the lateral primordia or bordering the newly established meristematic region, suggesting a more general role of PLA1 in signaling between indeterminate and determinate cells during the formation of lateral organs. Spatial transcriptomics builds on RNA ISH, which assays relatively few transcripts at a time and provides a powerful complement to single-cell transcriptomics that inherently removes cells from their native spatial context. Further improvements in resolution and sensitivity will greatly advance research in plant developmental biology.

Using single-plant-omics in the field to link maize genes to functions and phenotypes.

Most of our current knowledge on plant molecular biology is based on experiments in controlled laboratory environments. However, translating this knowledge from the laboratory to the field is often not straightforward, in part because field growth conditions are very different from laboratory conditions. Here, we test a new experimental design to unravel the molecular wiring of plants and study gene-phenotype relationships directly in the field. We molecularly profiled a set of individual maize plants of the same inbred background grown in the same field and used the resulting data to predict the phenotypes of individual plants and the function of maize genes. We show that the field transcriptomes of individual plants contain as much information on maize gene function as traditional laboratory-generated transcriptomes of pooled plant samples subject to controlled perturbations. Moreover, we show that field-generated transcriptome and metabolome data can be used to quantitatively predict individual plant phenotypes. Our results show that profiling individual plants in the field is a promising experimental design that could help narrow the lab-field gap.

Altered expression of maize PLASTOCHRON1 enhances biomass and seed yield by extending cell division duration.

Maize is the highest yielding cereal crop grown worldwide for grain or silage. Here, we show that modulating the expression of the maize PLASTOCHRON1 (ZmPLA1) gene, encoding a cytochrome P450 (CYP78A1), results in increased organ growth, seedling vigour, stover biomass and seed yield. The engineered trait is robust as it improves yield in an inbred as well as in a panel of hybrids, at several locations and over multiple seasons in the field. Transcriptome studies, hormone measurements and the expression of the auxin responsive DR5rev:mRFPer marker suggest that PLA1 may function through an increase in auxin. Detailed analysis of growth over time demonstrates that PLA1 stimulates the duration of leaf elongation by maintaining dividing cells in a proliferative, undifferentiated state for a longer period of time. The prolonged duration of growth also compensates for growth rate reduction caused by abiotic stresses.

A Conserved Core of Programmed Cell Death Indicator Genes Discriminates Developmentally and Environmentally Induced Programmed Cell Death in Plants.

A plethora of diverse programmed cell death (PCD) processes has been described in living organisms. In animals and plants, different forms of PCD play crucial roles in development, immunity, and responses to the environment. While the molecular control of some animal PCD forms such as apoptosis is known in great detail, we still know comparatively little about the regulation of the diverse types of plant PCD. In part, this deficiency in molecular understanding is caused by the lack of reliable reporters to detect PCD processes. Here, we addressed this issue by using a combination of bioinformatics approaches to identify commonly regulated genes during diverse plant PCD processes in Arabidopsis (Arabidopsis thaliana). Our results indicate that the transcriptional signatures of developmentally controlled cell death are largely distinct from the ones associated with environmentally induced cell death. Moreover, different cases of developmental PCD share a set of cell death-associated genes. Most of these genes are evolutionary conserved within the green plant lineage, arguing for an evolutionary conserved core machinery of developmental PCD. Based on this information, we established an array of specific promoter-reporter lines for developmental PCD in Arabidopsis. These PCD indicators represent a powerful resource that can be used in addition to established morphological and biochemical methods to detect and analyze PCD processes in vivo and in planta.

Only in dying, life: programmed cell death during plant development.

Programmed cell death (PCD) is a fundamental process of life. During the evolution of multicellular organisms, the actively controlled demise of cells has been recruited to fulfil a multitude of functions in development, differentiation, tissue homeostasis, and immune systems. In this review we discuss some of the multiple cases of PCD that occur as integral parts of plant development in a remarkable variety of cell types, tissues, and organs. Although research in the last decade has discovered a number of PCD regulators, mediators, and executers, we are still only beginning to understand the mechanistic complexity that tightly controls preparation, initiation, and execution of PCD as a process that is indispensable for successful vegetative and reproductive development of plants.

Programmed cell death controlled by ANAC033/SOMBRERO determines root cap organ size in Arabidopsis.

BACKGROUND: The root cap is a plant organ that ensheathes the meristematic stem cells at the root tip. Unlike other plant organs, the root cap shows a rapid cellular turnover, balancing constant cell generation by specific stem cells with the disposal of differentiated cells at the root cap edge. This cellular turnover is critical for the maintenance of root cap size and its position around the growing root tip, but how this is achieved and controlled in the model plant Arabidopsis thaliana remains subject to contradictory hypotheses. RESULTS: Here, we show that a highly organized cell death program is the final step of lateral root cap differentiation and that preparation for cell death is transcriptionally controlled by ANAC033/SOMBRERO. Precise timing of cell death is critical for the elimination of root cap cells before they fully enter the root elongation zone, which in turn is important in order to allow optimal root growth. Root cap cell death is followed by a rapid cell-autonomous corpse clearance and DNA fragmentation dependent on the S1-P1 type nuclease BFN1. CONCLUSIONS: Based on these results, we propose a novel concept in plant development that recognizes programmed cell death as a mechanism for maintaining organ size and tissue homeostasis in the Arabidopsis root cap.

Recent Progress in Development of Tnt1 Functional Genomics Platform for Medicago truncatula and Lotus japonicus in Bulgaria.

Legumes, as protein-rich crops, are widely used for human food, animal feed and vegetable oil production. Over the past decade, two legume species, Medicago truncatula and Lotus japonicus, have been adopted as model legumes for genomics and physiological studies. The tobacco transposable element, Tnt1, is a powerful tool for insertional mutagenesis and gene inactivation in plants. A large collection of Tnt1-tagged lines of M. truncatula cv. Jemalong was generated during the course of the project 'GLIP': Grain Legumes Integrated Project, funded by the European Union (www.eugrainlegumes.org). In the project 'IFCOSMO': Integrated Functional and COmparative genomics Studies on the MOdel Legumes Medicago truncatula and Lotus japonicus, supported by a grant from the Ministry of Education, Youth and Science, Bulgaria, these lines are used for development of functional genomics platform of legumes in Bulgaria. This review presents recent advances in the evaluation of the M. truncatula Tnt1 mutant collection and outlines the steps that are taken in using the Tnt1-tagging for generation of a mutant collection of the second model legume L. japonicus. Both collections will provide a number of legume-specific mutants and serve as a resource for functional and comparative genomics research on legumes. Genomics technologies are expected to advance genetics and breeding of important legume crops (pea, faba bean, alfalfa and clover) in Bulgaria and worldwide.