PpSCARECROW1 (PpSCR1) regulates leaf blade and mid-vein development in Physcomitrium patens.

In plants, asymmetric cell divisions result in distinct cell fates forming large and small daughter cells, adding to the cellular diversity in an organ. SCARECROW (SCR), a GRAS domain-containing transcription factor controls asymmetric periclinal cell divisions in flowering plants by governing radial patterning of ground tissue in roots and cell proliferation in leaves. Though SCR homologs are present across land plant lineages, the current understanding of their role in cellular patterning and leaf development is mostly limited to flowering plants. Our phylogenetic analysis identified three SCR homologs in moss Physcomitrium patens, amongst which PpSCR1 showed highest expression in gametophores and its promoter activity was prominent at the mid-vein and the flanking leaf blade cells pointing towards its role in leaf development. Notably, out of the three SCR homologs, only the ppscr1 knock-out lines developed slender leaves with four times narrower leaf blade and three times thicker mid-vein. Detailed histology studies revealed that slender leaf phenotype is either due to the loss of anticlinal cell divisions or failure of periclinal division suppression in the leaf blade. RNA-Seq analyses revealed that genes responsible for cell division and differentiation are expressed differentially in the mutant. PpSCR1 overexpression lines exhibited significantly wider leaf lamina, further reconfirming the role in leaf development. Together, our data suggests that PpSCR1 is involved in the leaf blade and mid-vein development of moss and that its role in the regulation of cell division and proliferation is ancient and conserved among flowering plants and mosses.

Physcomitrium patens response to elevated CO2 is flexible and determined by an interaction between sugar and nitrogen availability.

Mosses hold a unique position in plant evolution and are crucial for protecting natural, long-term carbon storage systems such as permafrost and bogs. Due to small stature, mosses grow close to the soil surface and are exposed to high levels of CO2 , produced by soil respiration. However, the impact of elevated CO2 (eCO2 ) levels on mosses remains underexplored. We determined the growth responses of the moss Physcomitrium patens to eCO2 in combination with different nitrogen levels and characterized the underlying physiological and metabolic changes. Three distinct growth characteristics, an early transition to caulonema, the development of longer, highly pigmented rhizoids, and increased biomass, define the phenotypic responses of P. patens to eCO2 . Elevated CO2 impacts growth by enhancing the level of a sugar signaling metabolite, T6P. The quantity and form of nitrogen source influences these metabolic and phenotypic changes. Under eCO2 , P. patens exhibits a diffused growth pattern in the presence of nitrate, but ammonium supplementation results in dense growth with tall gametophores, demonstrating high phenotypic plasticity under different environments. These results provide a framework for comparing the eCO2 responses of P. patens with other plant groups and provide crucial insights into moss growth that may benefit climate change models.

Peptides from conserved tandem direct repeats of SHORT-LEAF regulate gametophore development in moss P. patens.

Tandem direct repeat (TDR)-containing proteins, present across all domains of life, play crucial roles in plant development and defense mechanisms. Previously, we identified that disruption of a bryophyte-specific protein family, SHORT-LEAF (SHLF), possessing the longest reported TDRs, is the cause of the shlf mutant phenotype in Physcomitrium patens. shlf exhibits reduced apical dominance, altered auxin distribution, and 2-fold shorter leaves. However, the molecular role of SHLF was unclear due to the absence of known conserved domains. Through a series of protein domain deletion analyses, here, we demonstrate the importance of the signal peptide and the conserved TDRs and report a minimal functional protein (miniSHLF) containing the N-terminal signal peptide and first two TDRs (N-TDR1-2). We also demonstrate that SHLF behaves as a secretory protein and that the TDRs contribute to a pool of secreted peptides essential for SHLF function. Further, we identified that the mutant secretome lacks SHLF peptides, which are abundant in WT and miniSHLF secretomes. Interestingly, shlf mutants supplemented with the secretome or peptidome from WT or miniSHLF showed complete or partial phenotypic recovery. Transcriptomic and metabolomic analyses revealed that shlf displays an elevated stress response, including high ROS activity and differential accumulation of genes and metabolites involved in the phenylpropanoid pathway, which may affect auxin distribution. The TDR-specific synthetic peptide SHLFpep3 (INIINAPLQGFKIA) also rescued the mutant phenotypes, including the altered auxin distribution, in a dosage-dependent manner and restored the mutant's stress levels. Our study shows that secretory SHLF peptides derived from conserved TDRs regulate moss gametophore development.

Moving beyond the arabidopsis-centric view of G-protein signaling in plants.

Heterotrimeric G-protein-mediated signaling is a key mechanism to transduce a multitude of endogenous and environmental signals in diverse organisms. The scope and expectations of plant G-protein research were set by pioneering work in metazoans. Given the similarity of the core constituents, G-protein-signaling mechanisms were presumed to be universally conserved. However, because of the enormous diversity of survival strategies and endless forms among eukaryotes, the signal, its interpretation, and responses vary even among different plant groups. Earlier G-protein research in arabidopsis (Arabidopsis thaliana) has emphasized its divergence from Metazoa. Here, we compare recent evidence from diverse plant lineages with the available arabidopsis G-protein model and discuss the conserved and novel protein components, signaling mechanisms, and response regulation.

The plant response to high CO2 levels is heritable and orchestrated by DNA methylation.

Plant responses to abiotic environmental challenges are known to have lasting effects on the plant beyond the initial stress exposure. Some of these lasting effects are transgenerational, affecting the next generation. The plant response to elevated carbon dioxide (CO2 ) levels has been well studied. However, these investigations are typically limited to plants grown for a single generation in a high CO2 environment while transgenerational studies are rare. We aimed to determine transgenerational growth responses in plants after exposure to high CO2 by investigating the direct progeny when returned to baseline CO2 levels. We found that both the flowering plant Arabidopsis thaliana and seedless nonvascular plant Physcomitrium patens continue to display accelerated growth rates in the progeny of plants exposed to high CO2 . We used the model species Arabidopsis to dissect the molecular mechanism and found that DNA methylation pathways are necessary for heritability of this growth response. More specifically, the pathway of RNA-directed DNA methylation is required to initiate methylation and the proteins CMT2 and CMT3 are needed for the transgenerational propagation of this DNA methylation to the progeny plants. Together, these two DNA methylation pathways establish and then maintain a cellular memory to high CO2 exposure.

Abscisic acid-controlled redox proteome of Arabidopsis and its regulation by heterotrimeric Gß protein.

The plant hormone abscisic acid (ABA) plays crucial roles in regulation of stress responses and growth modulation. Heterotrimeric G-proteins are key mediators of ABA responses. Both ABA and G-proteins have also been implicated in intracellular redox regulation; however, the extent to which reversible protein oxidation manipulates ABA and/or G-protein signaling remains uncharacterized. To probe the role of reversible protein oxidation in plant stress response and its dependence on G-proteins, we determined the ABA-dependent reversible redoxome of wild-type and Gß-protein null mutant agb1 of Arabidopsis. We quantified 6891 uniquely oxidized cysteine-containing peptides, 923 of which show significant changes in oxidation following ABA treatment. The majority of these changes required the presence of G-proteins. Divergent pathways including primary metabolism, reactive oxygen species response, translation and photosynthesis exhibited both ABA- and G-protein-dependent redox changes, many of which occurred on proteins not previously linked to them. We report the most comprehensive ABA-dependent plant redoxome and uncover a complex network of reversible oxidations that allow ABA and G-proteins to rapidly adjust cellular signaling to adapt to changing environments. Physiological validation of a subset of these observations suggests that functional G-proteins are required to maintain intracellular redox homeostasis and fully execute plant stress responses.

Effect of environmental signals on growth and development in mosses.

Plants perceive a multitude of environmental signals and stresses, and integrate their response to them in ways that culminate in modified phenotypes, optimized for plant survival. This ability of plants, known as phenotypic plasticity, is found throughout evolution, in all plant lineages. For any given environment, the specifics of the response to a particular signal may vary depending on the plants' unique physiology and ecological niche. The bryophyte lineage, including mosses, which diverged from the vascular plants ~450-430 million years ago, represent a unique ecological and phylogenetic group in plant evolution. Several aspects of the moss life cycle, their morphology including the presence of specialized tissue types and distinct anatomical features, gene repertoires and networks, as well as the habitat differ significantly from those of vascular plants. To evaluate the outcomes of these differences, we explore the phenotypic responses of mosses to environmental signals such as light, temperature, CO2, water, nutrients, and gravity, and compare those with what is known in vascular plants. We also outline knowledge gaps and formulate testable hypotheses based on the contribution of anatomical and molecular factors to specific phenotypic responses.

Distribution and the evolutionary history of G-protein components in plant and algal lineages.

Heterotrimeric G-protein complexes comprising Gα-, Gß-, and Gγ-subunits and the regulator of G-protein signaling (RGS) are conserved across most eukaryotic lineages. Signaling pathways mediated by these proteins influence overall growth, development, and physiology. In plants, this protein complex has been characterized primarily from angiosperms with the exception of spreading-leaved earth moss (Physcomitrium patens) and Chara braunii (charophytic algae). Even within angiosperms, specific G-protein components are missing in certain species, whereas unique plant-specific variants-the extra-large Gα (XLGα) and the cysteine-rich Gγ proteins-also exist. The distribution and evolutionary history of G-proteins and their function in nonangiosperm lineages remain mostly unknown. We explored this using the wealth of available sequence data spanning algae to angiosperms representing extant species that diverged approximately 1,500 million years ago, using BLAST, synteny analysis, and custom-built Hidden Markov Model profile searches. We show that a minimal set of components forming the XLGαßγ trimer exists in the entire land plant lineage, but their presence is sporadic in algae. Additionally, individual components have distinct evolutionary histories. The XLGα exhibits many lineage-specific gene duplications, whereas Gα and RGS show several instances of gene loss. Similarly, Gß remained constant in both number and structure, but Gγ diverged before the emergence of land plants and underwent changes in protein domains, which led to three distinct subtypes. These results highlight the evolutionary oddities and summarize the phyletic patterns of this conserved signaling pathway in plants. They also provide a framework to formulate pertinent questions on plant G-protein signaling within an evolutionary context.

Evolutionarily Conserved and Non-Conserved Roles of Heterotrimeric Gα Proteins of Plants.

Heterotrimeric G-proteins modulate multiple signaling pathways in many eukaryotes. In plants, G-proteins have been characterized primarily from a few model angiosperms and a moss. Even within this small group, they seem to affect plant phenotypes differently: G-proteins are essential for survival in monocots, needed for adaptation but are nonessential in eudicots, and are required for life cycle completion and transition from the gametophytic to sporophytic phase in the moss Physcomitrium (Physcomitrella) patens. The classic G-protein heterotrimer consists of three subunits: one Gα, one Gß and one Gγ. The Gα protein is a catalytically active GTPase and, in its active conformation, interacts with downstream effectors to transduce signals. Gα proteins across the plant evolutionary lineage show a high degree of sequence conservation. To explore the extent to which this sequence conservation translates to their function, we complemented the well-characterized Arabidopsis Gα protein mutant, gpa1, with Gα proteins from different plant lineages and with the yeast Gpa1 and evaluated the transgenic plants for different phenotypes controlled by AtGPA1. Our results show that the Gα protein from a eudicot or a monocot, represented by Arabidopsis and Brachypodium, respectively, can fully complement all gpa1 phenotypes. However, the basal plant Gα failed to complement the developmental phenotypes exhibited by gpa1 mutants, although the phenotypes that are exhibited in response to various exogenous signals were partially or fully complemented by all Gα proteins. Our results offer a unique perspective on the evolutionarily conserved functions of G-proteins in plants.

The unique bryophyte-specific repeat-containing protein SHORT-LEAF regulates gametophore development in moss.

Convergent evolution of shoot development across plant lineages has prompted numerous comparative genetic studies. Though functional conservation of gene networks governing flowering plant shoot development has been explored in bryophyte gametophore development, the role of bryophyte-specific genes remains unknown. Previously, we have reported Tnt1 insertional mutants of moss defective in gametophore development. Here, we report a mutant (short-leaf; shlf) having two-fold shorter leaves, reduced apical dominance, and low plasmodesmata frequency. UHPLC-MS/MS-based auxin quantification and analysis of soybean (Glycine max) auxin-responsive promoter (GH3:GUS) lines exhibited a striking differential auxin distribution pattern in the mutant gametophore. Whole-genome sequencing and functional characterization of candidate genes revealed that a novel bryophyte-specific gene (SHORT-LEAF; SHLF) is responsible for the shlf phenotype. SHLF represents a unique family of near-perfect tandem direct repeat (TDR)-containing proteins conserved only among mosses and liverworts, as evident from our phylogenetic analysis. Cross-complementation with a Marchantia homolog partially recovered the shlf phenotype, indicating possible functional specialization. The distinctive structure (longest known TDRs), absence of any known conserved domain, localization in the endoplasmic reticulum, and proteolytic cleavage pattern of SHLF imply its function in bryophyte-specific cellular mechanisms. This makes SHLF a potential candidate to study gametophore development and evolutionary adaptations of early land plants.

Agrobacterium-mediated Tnt1 mutagenesis of moss protonemal filaments and generation of stable mutants with impaired gametophyte.

The gametophyte of moss exhibits a simple body plan, yet its growth is regulated by complex developmental phenomena similar to angiosperms. Because moss can be easily maintained under laboratory conditions, amenable for gene targeting and the availability of genome sequence, P. patens has become an attractive model system for studying evolutionary traits. Until date, there has been no Agrobacterium-mediated Tnt1 mutagenesis protocol for haploid protonemal filaments of moss. Hence, we attempted to use the intact tobacco Tnt1 retrotransposon as a mutagen for P. patens. Bioinformatic analysis of initiator methionyl-tRNA (Met-tRNAi), a critical host factor for Tnt1 transposition process, suggested that it can be explored as a mutagen for bryophytes. Using protonemal filaments and Agrobacterium-mediated transformation, 75 Tnt1 mutants have been generated and cryopreserved. SSAP analysis and TAIL-PCR revealed that Tnt1 is functional in P. patens and has a high-preference for gene and GC-rich regions. In addition, LTR::GUS lines exhibited a basal but tissue-specific inducible expression pattern. Forward genetic screen resulted in 5 novel phenotypes related to hormonal and gravity response, phyllid, and gamete development. SSAP analysis suggests that the Tnt1 insertion pattern is stable under normal growth conditions and the high-frequency phenotypic deviations are possibly due to the combination of haploid explant (protonema) and the choice of mutagen (Tnt1). We demonstrate that Agrobacterium-mediated Tnt1 insertional mutagenesis could generate stable P. patens mutant populations for future forward genetic studies.

Flower development, pollen fertility and sex expression analyses of three sexual phenotypes of Coccinia grandis.

BACKGROUND: Coccinia grandis is a dioecious species of Cucurbitaceae having heteromorphic sex chromosomes. The chromosome constitution of male and female plants is 22 + XY and 22 + XX respectively. Y chromosome of male sex is conspicuously large and plays a decisive role in determining maleness. Sex modification has been studied in hypogynous Silene latifolia (Caryophyllaceae) but there is no such report in epigynous Coccinia grandis. Moreover, the role of organ identity genes during sex expression in Coccinia has not been evaluated earlier. Investigations on sexual phenotypes of C. grandis including a rare gynomonoecious (GyM) form and AgNO3 mediated sex modification have added a new dimension to the understanding of sex expression in dioecious flowering plants. RESULTS: Morphometric analysis showed the presence of staminodes in pistillate flowers and histological study revealed the absence of carpel initials in male flowers. Though GyM plant had XX sex chromosomes, the development of stamens occurred in hermaphrodite flowers but the pollens were not fertile. Silver nitrate (AgNO3) application enhanced stamen growth in wild type female flowers like that of GyM plant but here also the pollens were sterile. Differential expression of CgPI could be involved in the development of different floral phenotypes. CONCLUSIONS: The three principle factors, Gynoecium Suppression (SuF), Stamen Promoting Factor (SPF) and Male Fertility (mF) that control sex expression in dioecious C. grandis assumed to be located on Y chromosome, play a decisive role in determining maleness. However, the characteristic development of stamens in hermaphrodite flowers of GyM plant having XX sex chromosomes indicates that Y-linked SPF regulatory pathway is somehow bypassed. Our experimental findings together with all other previous chromosomal and molecular cytogenetical data strongly support the view that C. grandis could be used as a potential model system to study sex expression in dioecious flowering plant.