Genome editing in macroalgae: advances and challenges.

This minireview examines the current state and challenges of genome editing in macroalgae. Despite the ecological and economic significance of this group of organisms, genome editing has seen limited applications. While CRISPR functionality has been established in two brown (Ectocarpus species 7 and Saccharina japonica) and one green seaweed (Ulva prolifera), these studies are limited to proof-of-concept demonstrations. All studies also (co)-targeted ADENINE PHOSPHORIBOSYL TRANSFERASE to enrich for mutants, due to the relatively low editing efficiencies. To advance the field, there should be a focus on advancing auxiliary technologies, particularly stable transformation, so that novel editing reagents can be screened for their efficiency. More work is also needed on understanding DNA repair in these organisms, as this is tightly linked with the editing outcomes. Developing efficient genome editing tools for macroalgae will unlock the ability to characterize their genes, which is largely uncharted terrain. Moreover, given their economic importance, genome editing will also impact breeding campaigns to develop strains that have better yields, produce more commercially valuable compounds, and show improved resilience to the impacts of global change.

Ulva: An emerging green seaweed model for systems biology.

Green seaweeds exhibit a wide range of morphologies and occupy various ecological niches, spanning from freshwater to marine and terrestrial habitats. These organisms, which predominantly belong to the class Ulvophyceae, showcase a remarkable instance of parallel evolution toward complex multicellularity and macroscopic thalli in the Viridiplantae lineage. Within the green seaweeds, several Ulva species ("sea lettuce") are model organisms for studying carbon assimilation, interactions with bacteria, life cycle progression, and morphogenesis. Ulva species are also notorious for their fast growth and capacity to dominate nutrient-rich, anthropogenically disturbed coastal ecosystems during "green tide" blooms. From an economic perspective, Ulva has garnered increasing attention as a promising feedstock for the production of food, feed, and biobased products, also as a means of removing excess nutrients from the environment. We propose that Ulva is poised to further develop as a model in green seaweed research. In this perspective, we focus explicitly on Ulva mutabilis/compressa as a model species and highlight the molecular data and tools that are currently available or in development. We discuss several areas that will benefit from future research or where exciting new developments have been reported in other Ulva species.

A Simple and Low-Tech Heat-Shock Method to Increase Genome Editing Efficiency in Plants.

CRISPR/Cas is now the standard technique to generate novel plant genotypes. However, optimizing the efficiency of the system continues to be an aspect of research and development. One of the improvements for increasing mutagenesis efficiency in different species is the application of heat stress. However, many experimental setups are limited by the requirement of using dedicated climate chambers to impose heat stress and by difficulties in the phenotyping of soil-grown plants. Here, we describe a simplified heat stress assay for in vitro-grown plants that can be completed in 6 days using commonly available laboratory equipment. We show that three 24-hr heat shocks (3×HS) at 37°C alternated with 24 hr of recovery at 21°C efficiently increases indel rates of LbCas12a and Cas9. We illustrate how visual mutant phenotypes (pds3 and gl1) can assist in quantifying genome editing efficiency, and describe how to quantify genome editing efficiency using genotyping by Sanger sequencing. We also provide a support protocol to efficiently clone a CRISPR expression vector in a single step. Together, our methods allow researchers to increase CRISPR-induced mutations using a low-tech setup in plants. © 2022 Wiley Periodicals LLC. Basic Protocol 1: 3×HS protocol Basic Protocol 2: Genotyping by Sanger sequencing Support Protocol: One-step cloning of a CRISPR expression vector.

The heat is on: a simple method to increase genome editing efficiency in plants.

BACKGROUND: Precision genome mutagenesis using CRISPR/Cas has become the standard method to generate mutant plant lines. Several improvements have been made to increase mutagenesis efficiency, either through vector optimisation or the application of heat stress. RESULTS: Here, we present a simplified heat stress assay that can be completed in six days using commonly-available laboratory equipment. We show that three heat shocks (3xHS) efficiently increases indel efficiency of LbCas12a and Cas9, irrespective of the target sequence or the promoter used to express the nuclease. The generated indels are primarily somatic, but for three out of five targets we demonstrate that up to 25% more biallelic mutations are transmitted to the progeny when heat is applied compared to non-heat controls. We also applied our heat treatment to lines containing CRISPR base editors and observed a 22-27% increase in the percentage of C-to-T base editing. Furthermore, we test the effect of 3xHS on generating large deletions and a homologous recombination reporter. Interestingly, we observed no positive effect of 3xHS treatment on either approach using our conditions. CONCLUSIONS: Together, our experiments show that heat treatment is consistently effective at increasing the number of somatic mutations using many CRISPR approaches in plants and in some cases can increase the recovery of mutant progeny.

Transcriptional dynamics of gametogenesis in the green seaweed Ulva mutabilis identifies an RWP-RK transcription factor linked to reproduction.

BACKGROUND: The molecular mechanism underlying sexual reproduction in land plants is well understood in model plants and is a target for crop improvement. However, unlike land plants, the genetic basis involved in triggering reproduction and gamete formation remains elusive in most seaweeds, which are increasingly viewed as an alternative source of functional food and feedstock for energy applications. RESULTS: Gametogenesis of Ulva mutabilis, a model organism for green seaweeds, was studied. We analyzed transcriptome dynamics at different time points during gametogenesis following induction of reproduction by fragmentation and removal of sporulation inhibitors. Analyses demonstrated that 45% of the genes in the genome were differentially expressed during gametogenesis. We identified several transcription factors that potentially play a key role in the early gametogenesis of Ulva given the function of their homologs in higher plants and microalgae. In particular, the detailed expression pattern of an evolutionarily conserved transcription factor containing an RWP-RK domain suggested a key role during Ulva gametogenesis. CONCLUSIONS: Transcriptomic analyses of gametogenesis in the green seaweed Ulva highlight the importance of a conserved RWP-RK transcription factor in the induction of sexual reproduction. The identification of putative master regulators of gametogenesis provides a starting point for further functional characterization.

Auxin's origin: do PILS hold the key?

Auxin is a key regulator of many developmental processes in land plants and plays a strikingly similar role in the phylogenetically distant brown seaweeds. Emerging evidence shows that the PIN and PIN-like (PILS) auxin transporter families have preceded the evolution of the canonical auxin response pathway. A wide conservation of PILS-mediated auxin transport, together with reports of auxin function in unicellular algae, would suggest that auxin function preceded the advent of multicellularity. We find that PIN and PILS transporters form two eukaryotic subfamilies within a larger bacterial family. We argue that future functional characterisation of algal PIN and PILS transporters can shed light on a common origin of an auxin function followed by independent co-option in a multicellular context.

A molecular toolkit for the green seaweed Ulva mutabilis.

The green seaweed Ulva mutabilis is an ecologically important marine primary producer as well as a promising cash crop cultivated for multiple uses. Despite its importance, several molecular tools are still needed to better understand seaweed biology. Here, we report the development of a flexible and modular molecular cloning toolkit for the green seaweed U. mutabilis based on a Golden Gate cloning system. The toolkit presently contains 125 entry vectors, 26 destination vectors, and 107 functionally validated expression vectors. We demonstrate the importance of endogenous regulatory sequences for transgene expression and characterize three endogenous promoters suitable to drive transgene expression. We describe two vector architectures to express transgenes via two expression cassettes or a bicistronic approach. The majority of selected transformants (50%-80%) consistently give clear visual transgene expression. Furthermore, we made different marker lines for intracellular compartments after evaluating 13 transit peptides and 11 tagged endogenous Ulva genes. Our molecular toolkit enables the study of Ulva gain-of-function lines and paves the way for gene characterization and large-scale functional genomics studies in a green seaweed.

Insights into the Evolution of Multicellularity from the Sea Lettuce Genome.

We report here the 98.5 Mbp haploid genome (12,924 protein coding genes) of Ulva mutabilis, a ubiquitous and iconic representative of the Ulvophyceae or green seaweeds. Ulva's rapid and abundant growth makes it a key contributor to coastal biogeochemical cycles; its role in marine sulfur cycles is particularly important because it produces high levels of dimethylsulfoniopropionate (DMSP), the main precursor of volatile dimethyl sulfide (DMS). Rapid growth makes Ulva attractive biomass feedstock but also increasingly a driver of nuisance "green tides." Ulvophytes are key to understanding the evolution of multicellularity in the green lineage, and Ulva morphogenesis is dependent on bacterial signals, making it an important species with which to study cross-kingdom communication. Our sequenced genome informs these aspects of ulvophyte cell biology, physiology, and ecology. Gene family expansions associated with multicellularity are distinct from those of freshwater algae. Candidate genes, including some that arose following horizontal gene transfer from chromalveolates, are present for the transport and metabolism of DMSP. The Ulva genome offers, therefore, new opportunities to understand coastal and marine ecosystems and the fundamental evolution of the green lineage.

The Mitochondrial DNA-Associated Protein SWIB5 Influences mtDNA Architecture and Homologous Recombination.

In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana Gain- and loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development.

F-Box Protein FBX92 Affects Leaf Size in Arabidopsis thaliana.

F-box proteins are part of one of the largest families of regulatory proteins that play important roles in protein degradation. In plants, F-box proteins are functionally very diverse, and only a small subset has been characterized in detail. Here, we identified a novel F-box protein FBX92 as a repressor of leaf growth in Arabidopsis. Overexpression of AtFBX92 resulted in plants with smaller leaves than the wild type, whereas plants with reduced levels of AtFBX92 showed, in contrast, increased leaf growth by stimulating cell proliferation. Detailed cellular analysis suggested that AtFBX92 specifically affects the rate of cell division during early leaf development. This is supported by the increased expression levels of several cell cycle genes in plants with reduced AtFBX92 levels. Surprisingly, overexpression of the maize homologous gene ZmFBX92 in maize had no effect on plant growth, whereas ectopic expression in Arabidopsis increased leaf growth. Expression of a truncated form of AtFBX92 showed that the contrasting effects of ZmFBX92 and AtFBX92 gain of function in Arabidopsis are due to the absence of the F-box-associated domain in the ZmFBX92 gene. Our work reveals an additional player in the complex network that determines leaf size and lays the foundation for identifying putative substrates.

Natural Variation of Molecular and Morphological Gibberellin Responses.

Although phytohormones such as gibberellins are essential for many conserved aspects of plant physiology and development, plants vary greatly in their responses to these regulatory compounds. Here, we use genetic perturbation of endogenous gibberellin levels to probe the extent of intraspecific variation in gibberellin responses in natural accessions of Arabidopsis (Arabidopsis thaliana). We find that these accessions vary greatly in their ability to buffer the effects of overexpression of GA20ox1, encoding a rate-limiting enzyme for gibberellin biosynthesis, with substantial differences in bioactive gibberellin concentrations as well as transcriptomes and growth trajectories. These findings demonstrate a surprising level of flexibility in the wiring of regulatory networks underlying hormone metabolism and signaling.

Plants grow with a little help from their organelle friends.

Chloroplasts and mitochondria are indispensable for plant development. They not only provide energy and carbon sources to cells, but also have evolved to become major players in a variety of processes such as amino acid metabolism, hormone biosynthesis and cellular signalling. As semi-autonomous organelles, they contain a small genome that relies largely on nuclear factors for its maintenance and expression. An intensive crosstalk between the nucleus and the organelles is therefore essential to ensure proper functioning, and the nuclear genes encoding organellar proteins involved in photosynthesis and oxidative phosphorylation are obviously crucial for plant growth. Organ growth is determined by two main cellular processes: cell proliferation and cell expansion. Here, we review how plant growth is affected in mutants of organellar proteins that are differentially expressed during leaf and root development. Our findings indicate a clear role for organellar proteins in plant organ growth, primarily during cell proliferation. However, to date, the role of the nuclear-encoded organellar proteins in the cellular processes driving organ growth has not been investigated in much detail. We therefore encourage researchers to extend their phenotypic characterization beyond macroscopic features in order to get a better view on how chloroplasts and mitochondria regulate the basic processes of cell proliferation and cell expansion, essential to driving growth.

Functional characterization of the Arabidopsis transcription factor bZIP29 reveals its role in leaf and root development.

Plant bZIP group I transcription factors have been reported mainly for their role during vascular development and osmosensory responses. Interestingly, bZIP29 has been identified in a cell cycle interactome, indicating additional functions of bZIP29 in plant development. Here, bZIP29 was functionally characterized to study its role during plant development. It is not present in vascular tissue but is specifically expressed in proliferative tissues. Genome-wide mapping of bZIP29 target genes confirmed its role in stress and osmosensory responses, but also identified specific binding to several core cell cycle genes and to genes involved in cell wall organization. bZIP29 protein complex analyses validated interaction with other bZIP group I members and provided insight into regulatory mechanisms acting on bZIP dimers. In agreement with bZIP29 expression in proliferative tissues and with its binding to promoters of cell cycle regulators, dominant-negative repression of bZIP29 altered the cell number in leaves and in the root meristem. A transcriptome analysis on the root meristem, however, indicated that bZIP29 might regulate cell number through control of cell wall organization. Finally, ectopic dominant-negative repression of bZIP29 and redundant factors led to a seedling-lethal phenotype, pointing to essential roles for bZIP group I factors early in plant development.

High-resolution time-resolved imaging of in vitro Arabidopsis rosette growth.

Although quantitative characterization of growth phenotypes is of key importance for the understanding of essential networks driving plant growth, the majority of growth-related genes are still being identified based on qualitative visual observations and/or single-endpoint quantitative measurements. We developed an in vitro growth imaging system (IGIS) to perform time-resolved analysis of rosette growth. In this system, Arabidopsis plants are grown in Petri dishes mounted on a rotating disk, and images of each plate are taken on an hourly basis. Automated image analysis was developed in order to obtain several growth-related parameters, such as projected rosette area, rosette relative growth rate, compactness and stockiness, over time. To illustrate the use of the platform and the resulting data, we present the results for the growth response of Col-0 plants subjected to three mild stress conditions. Although the reduction in rosette area was relatively similar at 19 days after stratification, the time-lapse analysis demonstrated that plants react differently to salt, osmotic and oxidative stress. The rosette area was altered at various time points during development, and leaf movement and shape parameters were also affected differently. We also used the IGIS to analyze in detail the growth behavior of mutants with enhanced leaf size. Analysis of several growth-related parameters over time in these mutants revealed several specificities in growth behavior, underlining the high complexity of leaf growth coordination. These results demonstrate that time-resolved imaging of in vitro rosette growth generates a better understanding of growth phenotypes than endpoint measurements.

The cell-cycle interactome: a source of growth regulators?

When plants develop, cell proliferation and cell expansion are tightly controlled in order to generate organs with a determinate final size such as leaves. Several studies have demonstrated the importance of the cell proliferation phase for leaf growth, illustrating that cell-cycle regulation is crucial for correct leaf development. A large and complex set of interacting proteins that constitute the cell-cycle interactome controls the transition from one cell-cycle phase to another. Here, we review the current knowledge on cell-cycle regulators from this interactome affecting final leaf size when their expression is altered, mainly in Arabidopsis. In addition to the description of mutants of CYCLIN-DEPENDENT KINASES (CDKs), CYCLINS (CYCs), and their transcriptional and post-translational regulators, a phenotypic analysis of gain- and loss-of-function mutants for 27 genes encoding proteins that interact with cell-cycle proteins is presented. This compilation of information shows that when cell-cycle-related genes are mis-expressed, leaf growth is often altered and that, seemingly, three main trends appear to be crucial in the regulation of final organ size by cell-cycle-related genes: (i) cellular compensation; (ii) gene dosage; and (iii) correct transition through the G2/M phase by ANAPHASE PROMOTING COMPLEX/CYCLOSOME (APC/C) activation. In conclusion, this meta-analysis shows that the cell-cycle interactome is enriched in leaf growth regulators, and illustrates the potential to identify new leaf growth regulators among putative new cell-cycle regulators.