Balancing growth amidst salt stress - lifestyle perspectives from the extremophyte model Schrenkiella parvula.

Schrenkiella parvula, a leading extremophyte model in Brassicaceae, can grow and complete its lifecycle under multiple environmental stresses, including high salinity. Yet, the key physiological and structural traits underlying its stress-adapted lifestyle are unknown along with trade-offs when surviving salt stress at the expense of growth and reproduction. We aimed to identify the influential adaptive trait responses that lead to stress-resilient and uncompromised growth across developmental stages when treated with salt at levels known to inhibit growth in Arabidopsis and most crops. Its resilient growth was promoted by traits that synergistically allowed primary root growth in seedlings, the expansion of xylem vessels across the root-shoot continuum, and a high capacity to maintain tissue water levels by developing thicker succulent leaves while enabling photosynthesis during salt stress. A successful transition from vegetative to reproductive phase was initiated by salt-induced early flowering, resulting in viable seeds. Self-fertilization in salt-induced early flowering was dependent upon filament elongation in flowers otherwise aborted in the absence of salt during comparable plant ages. The maintenance of leaf water status promoting growth, and early flowering to ensure reproductive success in a changing environment, were among the most influential traits that contributed to the extremophytic lifestyle of S. parvula.

Transcription Factor PecS Mediates Agrobacterium fabrum Fitness and Survival.

The transcriptional regulator PecS is encoded by select bacterial pathogens. For instance, in the plant pathogen Dickeya dadantii, PecS controls a range of virulence genes, including pectinase genes and the divergently oriented gene pecM, which encodes an efflux pump through which the antioxidant indigoidine is exported. In the plant pathogen Agrobacterium fabrum (formerly named Agrobacterium tumefaciens), the pecS-pecM locus is conserved. Using a strain of A. fabrum in which pecS has been disrupted, we show here that PecS controls a range of phenotypes that are associated with bacterial fitness. PecS represses flagellar motility and chemotaxis, which are processes that are important for A. fabrum to reach plant wound sites. Biofilm formation and microaerobic survival are reduced in the pecS disruption strain, whereas the production of acyl homoserine lactone (AHL) and resistance to reactive oxygen species (ROS) are increased when pecS is disrupted. AHL production and resistance to ROS are expected to be particularly relevant in the host environment. We also show that PecS does not participate in the induction of vir genes. The inducing ligands for PecS, urate, and xanthine, may be found in the rhizosphere, and they accumulate within the plant host upon infection. Therefore, our data suggest that PecS mediates A. fabrum fitness during its transition from the rhizosphere to the host plant. IMPORTANCE PecS is a transcription factor that is conserved in several pathogenic bacteria, where it regulates virulence genes. The plant pathogen Agrobacterium fabrum is important not only for its induction of crown galls in susceptible plants but also for its role as a tool in the genetic manipulation of host plants. We show here that A. fabrum PecS controls a range of phenotypes, which would confer the bacteria an advantage while transitioning from the rhizosphere to the host plant. This includes the production of signaling molecules, which are critical for the propagation of the tumor-inducing plasmid. A more complete understanding of the infection process may inform approaches by which to treat infections as well as to facilitate the transformation of recalcitrant plant species.

The CDK Inhibitor SIAMESE Targets Both CDKA;1 and CDKB1 Complexes to Establish Endoreplication in Trichomes.

Endoreplication, also known as endoreduplication, is a modified cell cycle in which DNA is replicated without subsequent cell division. Endoreplication plays important roles in both normal plant development and in stress responses. The SIAMESE (SIM) gene of Arabidopsis (Arabidopsis thaliana) encodes a cyclin-dependent kinase (CDK) inhibitor that plays a central role in establishing endoreplication, and is the founding member of the SIAMESE-RELATED (SMR) family of plant-specific CDK inhibitor genes. However, there has been conflicting evidence regarding which specific cyclin/CDK complexes are inhibited by SIM in vivo. In this work, we use genetic evidence to show that SIM likely inhibits both CDKA;1- and CDKB1-containing CDK complexes in vivo, thus promoting endoreplication in developing Arabidopsis trichomes. We also show that SIM interacts with CYCA2;3, a binding partner of CDKB1;1, via SIM motif A, which we previously identified as a CDK-binding motif. By contrast, SIM motif C, which has been indicated as a cyclin binding motif in other contexts, appears to be relatively unimportant for interaction between SIM and CYCA2;3. Together with earlier results, our work suggests that SIM and other SMRs likely have a multivalent interaction with CYC/CDK complexes.

A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation.

Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing Arabidopsis thaliana root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments.

Functional Analysis of Short Linear Motifs in the Plant Cyclin-Dependent Kinase Inhibitor SIAMESE.

Endoreplication, a modified cell cycle in which DNA is replicated without subsequent cell division, plays an important but poorly understood role in plant growth and in plant responses to biotic and abiotic stress. The Arabidopsis (Arabidopsis thaliana) SIAMESE (SIM) gene encodes the first identified member of the SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors. SIM controls endoreplication during trichome development, and sim mutant trichomes divide several times instead of endoreplicating their DNA. The SMR family is defined by several short linear amino acid sequence motifs of largely unknown function, and family members have little sequence similarity to any known protein functional domains. Here, we investigated the roles of the conserved motifs in SIM site-directed Arabidopsis mutants using several functional assays. We identified a potential cyclin-dependent kinase (CDK)-binding site, which bears no resemblance to other known CDK interaction motifs. We also identified a potential site of phosphorylation and two redundant nuclear localization sequences. Surprisingly, the only motif with similarity to the other family of plant CDK inhibitors, the INHIBITOR/INTERACTOR OF CDC2 KINASE/KIP-RELATED PROTEIN proteins, is not required for SIM function in vivo. Because even highly divergent members of the SMR family are able to replace SIM function in Arabidopsis trichomes, it is likely that the results obtained here for SIM will apply to other members of this plant-specific family of CDK inhibitors.

Making Epidermal Bladder Cells Bigger: Developmental- and Salinity-Induced Endopolyploidy in a Model Halophyte.

Endopolyploidy occurs when DNA replication takes place without subsequent mitotic nuclear division, resulting in cell-specific ploidy levels within tissues. In plants, endopolyploidy plays an important role in sustaining growth and development, but only a few studies have demonstrated a role in abiotic stress response. In this study, we investigated the function of ploidy level and nuclear and cell size in leaf expansion throughout development and tracked cell type-specific ploidy in the halophyte Mesembryanthemum crystallinum In addition to developmental endopolyploidy, we examined the effects of salinity stress on ploidy level. We focused specifically on epidermal bladder cells (EBC), which are modified balloon-like trichomes, due to their large size and role in salt accumulation. Our results demonstrate that ploidy increases as the leaves expand in a similar manner for each leaf type, and ploidy levels up to 512C were recorded for nuclei in EBC of leaves of adult plants. Salt treatment led to a significant increase in ploidy levels in the EBC, and these cells showed spatially related differences in their ploidy and nuclear and cell size depending on the positions on the leaf and stem surface. Transcriptome analysis highlighted salinity-induced changes in genes involved in DNA replication, cell cycle, endoreduplication, and trichome development in EBC. The increase in cell size and ploidy observed in M. crystallinum under salinity stress may contribute to salt tolerance by increasing the storage capacity for sodium sequestration brought about by higher metabolic activity driving rapid cell enlargement in the leaf tissue and EBC.

Functional Conservation in the SIAMESE-RELATED Family of Cyclin-Dependent Kinase Inhibitors in Land Plants.

The best-characterized members of the plant-specific SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors regulate the transition from the mitotic cell cycle to endoreplication, also known as endoreduplication, an altered version of the cell cycle in which DNA is replicated without cell division. Some other family members are implicated in cell cycle responses to biotic and abiotic stresses. However, the functions of most SMRs remain unknown, and the specific cyclin-dependent kinase complexes inhibited by SMRs are unclear. Here, we demonstrate that a diverse group of SMRs, including an SMR from the bryophyte Physcomitrella patens, can complement an Arabidopsis thaliana siamese (sim) mutant and that both Arabidopsis SIM and P. patens SMR can inhibit CDK activity in vitro. Furthermore, we show that Arabidopsis SIM can bind to and inhibit both CDKA;1 and CDKB1;1. Finally, we show that SMR2 acts to restrict cell proliferation during leaf growth in Arabidopsis and that SIM, SMR1/LGO, and SMR2 play overlapping roles in controlling the transition from cell division to endoreplication during leaf development. These results indicate that differences in SMR function in plant growth and development are primarily due to differences in transcriptional and posttranscriptional regulation, rather than to differences in fundamental biochemical function.

Differential Roles of Two Homologous Cyclin-Dependent Kinase Inhibitor Genes in Regulating Cell Cycle and Innate Immunity in Arabidopsis.

Precise cell-cycle control is critical for plant development and responses to pathogen invasion. Two homologous cyclin-dependent kinase inhibitor genes, SIAMESE (SIM) and SIM-RELATED 1 (SMR1), were recently shown to regulate Arabidopsis (Arabidopsis thaliana) defense based on phenotypes conferred by a sim smr1 double mutant. However, whether these two genes play differential roles in cell-cycle and defense control is unknown. In this report, we show that while acting synergistically to promote endoreplication, SIM and SMR1 play different roles in affecting the ploidy of trichome and leaf cells, respectively. In addition, we found that the smr1-1 mutant, but not sim-1, was more susceptible to a virulent Pseudomonas syringae strain, and this susceptibility could be rescued by activating salicylic acid (SA)-mediated defense. Consistent with these results, smr1-1 partially suppressed the dwarfism, high SA levels, and cell death phenotypes in acd6-1, a mutant used to gauge the change of defense levels. Thus, SMR1 functions partly through SA in defense control. The differential roles of SIM and SMR1 are due to differences in temporal and spatial expression of these two genes in Arabidopsis tissues and in response to P. syringae infection. In addition, flow-cytometry analysis of plants with altered SA signaling revealed that SA is necessary, but not sufficient, to change cell-cycle progression. We further found that a mutant with three CYCD3 genes disrupted also compromised disease resistance to P. syringae. Together, this study reveals differential roles of two homologous cyclin-dependent kinase inhibitors in regulating cell-cycle progression and innate immunity in Arabidopsis and provides insights into the importance of cell-cycle control during host-pathogen interactions.

BRANCHLESS TRICHOMES links cell shape and cell cycle control in Arabidopsis trichomes.

Endoreplication, also called endoreduplication, is a modified cell cycle in which DNA is repeatedly replicated without subsequent cell division. Endoreplication is often associated with increased cell size and specialized cell shapes, but the mechanism coordinating DNA content with shape and size remains obscure. Here we identify the product of the BRANCHLESS TRICHOMES (BLT) gene, a protein of hitherto unknown function that has been conserved throughout angiosperm evolution, as a link in coordinating cell shape and nuclear DNA content in endoreplicated Arabidopsis trichomes. Loss-of-function mutations in BLT were found to enhance the multicellular trichome phenotype of mutants in the SIAMESE (SIM) gene, which encodes a repressor of endoreplication. Epistasis and overexpression experiments revealed that BLT encodes a key regulator of trichome branching. Additional experiments showed that BLT interacts both genetically and physically with STICHEL, another key regulator of trichome branching. Although blt mutants have normal trichome DNA content, overexpression of BLT results in an additional round of endoreplication, and blt mutants uncouple DNA content from morphogenesis in mutants with increased trichome branching, further emphasizing its role in linking cell shape and endoreplication.

Endoreplication controls cell fate maintenance.

Cell-fate specification is typically thought to precede and determine cell-cycle regulation during differentiation. Here we show that endoreplication, also known as endoreduplication, a specialized cell-cycle variant often associated with cell differentiation but also frequently occurring in malignant cells, plays a role in maintaining cell fate. For our study we have used Arabidopsis trichomes as a model system and have manipulated endoreplication levels via mutants of cell-cycle regulators and overexpression of cell-cycle inhibitors under a trichome-specific promoter. Strikingly, a reduction of endoreplication resulted in reduced trichome numbers and caused trichomes to lose their identity. Live observations of young Arabidopsis leaves revealed that dedifferentiating trichomes re-entered mitosis and were re-integrated into the epidermal pavement-cell layer, acquiring the typical characteristics of the surrounding epidermal cells. Conversely, when we promoted endoreplication in glabrous patterning mutants, trichome fate could be restored, demonstrating that endoreplication is an important determinant of cell identity. Our data lead to a new model of cell-fate control and tissue integrity during development by revealing a cell-fate quality control system at the tissue level.

Targeted interactomics reveals a complex core cell cycle machinery in Arabidopsis thaliana.

Cell proliferation is the main driving force for plant growth. Although genome sequence analysis revealed a high number of cell cycle genes in plants, little is known about the molecular complexes steering cell division. In a targeted proteomics approach, we mapped the core complex machinery at the heart of the Arabidopsis thaliana cell cycle control. Besides a central regulatory network of core complexes, we distinguished a peripheral network that links the core machinery to up- and downstream pathways. Over 100 new candidate cell cycle proteins were predicted and an in-depth biological interpretation demonstrated the hypothesis-generating power of the interaction data. The data set provided a comprehensive view on heterodimeric cyclin-dependent kinase (CDK)-cyclin complexes in plants. For the first time, inhibitory proteins of plant-specific B-type CDKs were discovered and the anaphase-promoting complex was characterized and extended. Important conclusions were that mitotic A- and B-type cyclins form complexes with the plant-specific B-type CDKs and not with CDKA;1, and that D-type cyclins and S-phase-specific A-type cyclins seem to be associated exclusively with CDKA;1. Furthermore, we could show that plants have evolved a combinatorial toolkit consisting of at least 92 different CDK-cyclin complex variants, which strongly underscores the functional diversification among the large family of cyclins and reflects the pivotal role of cell cycle regulation in the developmental plasticity of plants.

How do cells know what they want to be when they grow up? Lessons from epidermal patterning in Arabidopsis.

Because the plant epidermis is readily accessible and consists of few cell types on most organs, the epidermis has become a well-studied model for cell differentiation and cell patterning in plants. Recent advances in our understanding of the development of three epidermal cell types, trichomes, root hairs, and stomata, allow a comparison of the underlying patterning mechanisms. In Arabidopsis, trichome development and root epidermal patterning use a common mechanism involving closely related cell fate transcription factors and a similar lateral inhibition signaling pathway. Yet the resulting patterns differ substantially, primarily due to the influence of a prepattern derived from subepidermal cortical cells in root epidermal patterning. Stomatal patterning uses a contrasting mechanism based primarily on control of the orientation of cell divisions that also involves an inhibitory signaling pathway. This review focuses on comparing and contrasting these patterning pathways to identify and illustrate general themes that may be broadly applicable to other systems. Where these pathways occur in the same tissue, interaction and competition between these pathways is also discussed.

Constitutive Expressor Of Pathogenesis-related Genes5 affects cell wall biogenesis and trichome development.

BACKGROUND: The Arabidopsis thaliana CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATED GENES5 (CPR5) gene has been previously implicated in disease resistance, cell proliferation, cell death, and sugar sensing, and encodes a putative membrane protein of unknown biochemical function. Trichome development is also affected in cpr5 plants, which have leaf trichomes that are reduced in size and branch number. RESULTS: In the work presented here, the role of CPR5 in trichome development was examined. Trichomes on cpr5 mutants had reduced birefringence, suggesting a difference in cell wall structure between cpr5 and wild-type trichomes. Consistent with this, leaf cell walls of cpr5 plants contained significantly less paracrystalline cellulose and had an altered wall carbohydrate composition. We also found that the effects of cpr5 on trichome size and endoreplication of trichome nuclear DNA were epistatic to the effects of mutations in triptychon (try) or overexpression of GLABRA3, indicating that these trichome developmental regulators are dependant on CPR5 function for their effects on trichome expansion and endoreplication. CONCLUSION: Our results suggest that CPR5 is unlikely to be a specific regulator of pathogen response pathways or senescence, but rather functions either in cell wall biogenesis or in multiple cell signaling or transcription response pathways.

A Tale of Two Cycles.

Two of the fundamental rhythms of eukaryotic life are the circadian clock and the cell division cycle. In this issue of Developmental Cell, Fung-Uceda and colleagues (2018) have elucidated a molecular mechanism linking the circadian clock to the cell cycle in the plant Arabidopsis thaliana.

Making Plants Break a Sweat: the Structure, Function, and Evolution of Plant Salt Glands.

Salt stress is a complex trait that poses a grand challenge in developing new crops better adapted to saline environments. Some plants, called recretohalophytes, that have naturally evolved to secrete excess salts through salt glands, offer an underexplored genetic resource for examining how plant development, anatomy, and physiology integrate to prevent excess salt from building up to toxic levels in plant tissue. In this review we examine the structure and evolution of salt glands, salt gland-specific gene expression, and the possibility that all salt glands have originated via evolutionary modifications of trichomes. Salt secretion via salt glands is found in more than 50 species in 14 angiosperm families distributed in caryophyllales, asterids, rosids, and grasses. The salt glands of these distantly related clades can be grouped into four structural classes. Although salt glands appear to have originated independently at least 12 times, they share convergently evolved features that facilitate salt compartmentalization and excretion. We review the structural diversity and evolution of salt glands, major transporters and proteins associated with salt transport and secretion in halophytes, salt gland relevant gene expression regulation, and the prospect for using new genomic and transcriptomic tools in combination with information from model organisms to better understand how salt glands contribute to salt tolerance. Finally, we consider the prospects for using this knowledge to engineer salt glands to increase salt tolerance in model species, and ultimately in crops.

Corrigendum: Making Plants Break a Sweat: the Structure, Function, and Evolution of Plant Salt Glands.

[This corrects the article on p. 406 in vol. 8, PMID: 28400779.].

Why do plants need so many cyclin-dependent kinase inhibitors?

Cell cycle regulation is fundamental to growth and development, and Cyclin-Dependent Kinase Inhibitors (CKIs) are major negative regulators of the cell cycle. Plant genomes encode substantially more CKIs than metazoan or fungal genomes. Plant CKIs fall into 2 distinct families, KIP-RELATED PROTEINS (KRPs) and SIAMESE-RELATED proteins (SMRs). SMRs can inhibit both S-phase and M-phase CDK complexes in vitro and are transcribed throughout the cell cycle, yet SMRs do not inhibit DNA replication in vivo. This suggests that SMRs must be activated post transcriptionally after the start of S-phase, but the mechanism of this hypothesized activation is unknown. Recent work indicates that even distantly related SMRs have the same biochemical function, and that differential transcriptional regulation likely maintains their distinct roles in integrating various environmental and developmental signals with the cell cycle.

Mechanical Shielding of Rapidly Growing Cells Buffers Growth Heterogeneity and Contributes to Organ Shape Reproducibility.

A landmark of developmental biology is the production of reproducible shapes, through stereotyped morphogenetic events. At the cell level, growth is often highly heterogeneous, allowing shape diversity to arise. Yet, how can reproducible shapes emerge from such growth heterogeneity? Is growth heterogeneity filtered out? Here, we focus on rapidly growing trichome cells in the Arabidopsis sepal, a reproducible floral organ. We show via computational modeling that rapidly growing cells may distort organ shape. However, the cortical microtubule alignment along growth-derived maximal tensile stress in adjacent cells would mechanically isolate rapidly growing cells and limit their impact on organ shape. In vivo, we observed such microtubule response to stress and consistently found no significant effect of trichome number on sepal shape in wild-type and lines with trichome number defects. Conversely, modulating the microtubule response to stress in katanin and spiral2 mutant made sepal shape dependent on trichome number, suggesting that, while mechanical signals are propagated around rapidly growing cells, the resistance to stress in adjacent cells mechanically isolates rapidly growing cells, thus contributing to organ shape reproducibility.

Estimating the degree of saturation in mutant screens.

Large-scale screens for loss-of-function mutants have played a significant role in recent advances in developmental biology and other fields. In such mutant screens, it is desirable to estimate the degree of "saturation" of the screen (i.e., what fraction of the possible target genes has been identified). We applied Bayesian and maximum-likelihood methods for estimating the number of loci remaining undetected in large-scale screens and produced credibility intervals to assess the uncertainty of these estimates. Since different loci may mutate to alleles with detectable phenotypes at different rates, we also incorporated variation in the degree of mutability among genes, using either gamma-distributed mutation rates or multiple discrete mutation rate classes. We examined eight published data sets from large-scale mutant screens and found that credibility intervals are much broader than implied by previous assumptions about the degree of saturation of screens. The likelihood methods presented here are a significantly better fit to data from published experiments than estimates based on the Poisson distribution, which implicitly assumes a single mutation rate for all loci. The results are reasonably robust to different models of variation in the mutability of genes. We tested our methods against mutant allele data from a region of the Drosophila melanogaster genome for which there is an independent genomics-based estimate of the number of undetected loci and found that the number of such loci falls within the predicted credibility interval for our models. The methods we have developed may also be useful for estimating the degree of saturation in other types of genetic screens in addition to classical screens for simple loss-of-function mutants, including genetic modifier screens and screens for protein-protein interactions using the yeast two-hybrid method.

Molecular control and function of endoreplication in development and physiology.

Endoreplication, also called endoreduplication, is a cell cycle variant of multicellular eukaryotes in which mitosis is skipped and cells repeatedly replicate their DNA, resulting in cellular polyploidy. In recent years, research results have shed light on the molecular mechanism of endoreplication control, but the function of this cell-cycle variant has remained elusive. However, new evidence is at last providing insight into the biological relevance of cellular polyploidy, demonstrating that endoreplication is essential for developmental processes, such as cell fate maintenance, and is a prominent response to physiological conditions, such as pathogen attack or DNA damage. Thus, endoreplication is being revealed as an important module in plant growth that contributes to the robustness of plant life.