Malondialdehyde Assays in Higher Plants.

Malondialdehyde is a three-carbon dialdehyde produced as a byproduct of polyunsaturated fatty acid peroxidation widely used as a marker of the extent of lipid peroxidation in plants. There are several methodological approaches to quantify malondialdehyde contents in higher plants, ranging from the simplest, cheapest, and quickest spectrophotometric approaches to the more complex ones using tandem mass spectrometry. This chapter summarizes the advantages and limitations of approaches followed and provides brief protocols with some tips to facilitate the selection of the best method for each experimental condition and application.

Light Response Curves in Land Plants.

Light is the driving force for photosynthesis. Two techniques are commonly employed to help characterize the relationship between the light environment and photosynthesis in plants.Chlorophyll a fluorescence analysis is used to examine both the capacity for and the efficiency of the conversion of absorbed light into energy for photosynthesis. Additionally, gas exchange analysis is used to assess the utilization of that energy for carbon fixation. These techniques are used either in isolation or in combination to acquire light response curves that measure the response of the plant to sequential changes in irradiance. Light response curves can help users understand photosynthetic mechanisms, evaluate how plants respond to light conditions, or assess the extent of physiological plasticity within plants. In this chapter, we provide a generalized method for acquiring light response curves suitable for both chlorophyll a fluorescence and gas exchange techniques using commercially available apparatus. Depending on the equipment available, these methods can be applied individually or combined to acquire data simultaneously. The methods are broadly applicable to most land plants but are ideally suited to help those that are unfamiliar with these techniques.

Photosynthetic Gas Exchange in Land Plants at the Leaf Level.

Leaf-level gas exchange enables insights into the physiology and in vivo biochemical processes of plants. Advances in infrared gas analysis have resulted in user-friendly off-the-shelf gas exchange systems that allow researchers to collect physiological measurements with the push of a few buttons. Here, I describe how to set up the gas exchange equipment, what to pay attention to while making measurements, and provide some guidelines on how to analyze and interpret the data obtained.

Diversification of Plastid Structure and Function in Land Plants.

Plastids represent a largely diverse group of organelles in plant and algal cells that have several common features but also a broad spectrum of morphological, ultrastructural, biochemical, and physiological differences. Plastids and their structural and metabolic diversity significantly contribute to the functionality and developmental flexibility of the plant body throughout its lifetime. In addition to the multiple roles of given plastid types, this diversity is accomplished in some cases by interconversions between different plastids as a consequence of developmental and environmental signals that regulate plastid differentiation and specialization. In addition to basic plastid structural features, the most important plastid types, the newly characterized peculiar plastids, and future perspectives in plastid biology are also provided in this chapter.

Sexual reproduction: Is the genetic pathway for female germ cell specification conserved in land plants?

Land plants share several core factors responsible for female gametophyte development, despite their differing structures and developmental programs. New work providing molecular dissection of reproductive phases in non-angiosperm plants is a powerful tool for elucidating the underlying genetic network.

Plant evolution: Streptophyte multicellularity, ecology, and the acclimatisation of plants to life on land.

Land plants are celebrated as one of the three great instances of complex multicellularity, but new phylogenomic and phenotypic analyses are revealing deep evolutionary roots of multicellularity among algal relatives, prompting questions about the causal basis of this major evolutionary transition.

Cryogenian Origins of Multicellularity in Archaeplastida.

Earth was impacted by global glaciations during the Cryogenian (720 to 635 million years ago; Ma), events invoked to explain both the origins of multicellularity in Archaeplastida and radiation of the first land plants. However, the temporal relationship between these environmental and biological events is poorly established, due to a paucity of molecular and fossil data, precluding resolution of the phylogeny and timescale of archaeplastid evolution. We infer a time-calibrated phylogeny of early archaeplastid evolution based on a revised molecular dataset and reappraisal of the fossil record. Phylogenetic topology testing resolves deep archaeplastid relationships, identifying two clades of Viridiplantae and placing Bryopsidales as sister to the Chlorophyceae. Our molecular clock analysis infers an origin of Archaeplastida in the late-Paleoproterozoic to early-Mesoproterozoic (1712 to 1387 Ma). Ancestral state reconstruction of cytomorphological traits on this time-calibrated tree reveals many of the independent origins of multicellularity span the Cryogenian, consistent with the Cryogenian multicellularity hypothesis. Multicellular rhodophytes emerged 902 to 655 Ma while crown-Anydrophyta (Zygnematophyceae and Embryophyta) originated 796 to 671 Ma, broadly compatible with the Cryogenian plant terrestrialization hypothesis. Our analyses resolve the timetree of Archaeplastida with age estimates for ancestral multicellular archaeplastids coinciding with the Cryogenian, compatible with hypotheses that propose a role of Snowball Earth in plant evolution.

Plant evolution: A tapetum is now effectively present in all land plant lineages.

The tapetum, a tissue that elsewhere ensures correct spore development, is missing in some bryophytes. A new study shows that, in the liverwort, Marchantia polymorpha, a gene controlling spore wall deposition is expressed in the capsule lining, so these cells essentially function as a tapetum.

Research Progress of Group II Intron Splicing Factors in Land Plant Mitochondria.

Mitochondria are important organelles that provide energy for the life of cells. Group II introns are usually found in the mitochondrial genes of land plants. Correct splicing of group II introns is critical to mitochondrial gene expression, mitochondrial biological function, and plant growth and development. Ancestral group II introns are self-splicing ribozymes that can catalyze their own removal from pre-RNAs, while group II introns in land plant mitochondria went through degenerations in RNA structures, and thus they lost the ability to self-splice. Instead, splicing of these introns in the mitochondria of land plants is promoted by nuclear- and mitochondrial-encoded proteins. Many proteins involved in mitochondrial group II intron splicing have been characterized in land plants to date. Here, we present a summary of research progress on mitochondrial group II intron splicing in land plants, with a major focus on protein splicing factors and their probable functions on the splicing of mitochondrial group II introns.

The ALOG domain defines a family of plant-specific transcription factors acting during Arabidopsis flower development.

The ALOG (Arabidopsis LIGHT-DEPENDENT SHORT HYPOCOTYLS 1 (LSH1) and Oryza G1) proteins are conserved plant-specific Transcription Factors (TFs). They play critical roles in the development of various plant organs (meristems, inflorescences, floral organs, and nodules) from bryophytes to higher flowering plants. Despite the fact that the first members of this family were originally discovered in Arabidopsis, their role in this model plant has remained poorly characterized. Moreover, how these transcriptional regulators work at the molecular level is unknown. Here, we study the redundant function of the ALOG proteins LSH1,3,4 from Arabidopsis. We uncover their role in the repression of bract development and position them within a gene regulatory network controlling this process and involving the floral regulators LEAFY, BLADE-ON-PETIOLE, and PUCHI. Next, using in vitro genome-wide studies, we identified the conserved DNA motif bound by ALOG proteins from evolutionarily distant species (the liverwort Marchantia polymorpha and the flowering plants Arabidopsis, tomato, and rice). Resolution of the crystallographic structure of the ALOG DNA-binding domain in complex with DNA revealed the domain is a four-helix bundle with a disordered NLS and a zinc ribbon insertion between helices 2 and 3. The majority of DNA interactions are mediated by specific contacts made by the third alpha helix and the NLS. Taken together, this work provides the biochemical and structural basis for DNA-binding specificity of an evolutionarily conserved TF family and reveals its role as a key player in Arabidopsis flower development.

A subclass II bHLH transcription factor in Marchantia polymorpha gives insight into the ancestral land plant trait of spore formation.

Sporopollenin is often said to be one of the toughest biopolymers known to man. The shift in dormancy cell wall deposition from around the diploid zygotes of charophycean algae to sporopollenin around the haploid spores of land plants essentially imparted onto land plants the gift of passive motility, a key acquisition that contributed to their vast and successful colonization across terrestrial habitats.1,2 A putative transcription factor controlling the land plant mode of sporopollenin deposition is the subclass II bHLHs, which are conserved and novel to land plants, with mutants of genes in angiosperms and mosses divulging roles relating to tapetum degeneration and spore development.3,4,5,6,7 We demonstrate that a subclass II bHLH gene, MpbHLH37, regulates sporopollenin biosynthesis and deposition in the model liverwort Marchantia polymorpha. Mpbhlh37 sporophytes show a striking loss of secondary wall deposits of the capsule wall, the elaters, and the spore exine, all while maintaining spore viability, identifying MpbHLH37 as a master regulator of secondary wall deposits of the sporophyte. Localization of MpbHLH37 to the capsule wall and elaters of the sporophyte directly designates these tissue types as a bona fide tapetum in liverworts, giving support to the notion that the presence of a tapetum is an ancestral land plant trait. Finally, as early land plant spore walls exhibit evidence of tapetal deposition,8,9,10,11,12 a tapetal capsule wall could have provided these plants with a developmental mechanism for sporopollenin deposition.

d-amino acids metabolism reflects the evolutionary origin of higher plants and their adaptation to the environment.

d-amino acids are the d stereoisomers of the common l-amino acids found in proteins. Over the past two decades, the occurrence of d-amino acids in plants has been reported and circumstantial evidence for a role in various processes, including interaction with soil microorganisms or interference with cellular signalling, has been provided. However, examples are not numerous and d-amino acids can also be detrimental, some of them inhibiting growth and development. Thus, the persistence of d-amino acid metabolism in plants is rather surprising, and the evolutionary origins of d-amino acid metabolism are currently unclear. Systemic analysis of sequences associated with d-amino acid metabolism enzymes shows that they are not simply inherited from cyanobacterial metabolism. In fact, the history of plant d-amino acid metabolism enzymes likely involves multiple steps, cellular compartments, gene transfers and losses. Regardless of evolutionary steps, enzymes of d-amino acid metabolism, such as d-amino acid transferases or racemases, have been retained by higher plants and have not simply been eliminated, so it is likely that they fulfil important metabolic roles such as serine, folate or plastid peptidoglycan metabolism. We suggest that d-amino acid metabolism may have been critical to support metabolic functions required during the evolution of land plants.

Phylogenomic insights into the first multicellular streptophyte.

Streptophytes are best known as the clade containing the teeming diversity of embryophytes (land plants).1,2,3,4 Next to embryophytes are however a range of freshwater and terrestrial algae that bear important information on the emergence of key traits of land plants. Among these, the Klebsormidiophyceae stand out. Thriving in diverse environments-from mundane (ubiquitous occurrence on tree barks and rocks) to extreme (from the Atacama Desert to the Antarctic)-Klebsormidiophyceae can exhibit filamentous body plans and display remarkable resilience as colonizers of terrestrial habitats.5,6 Currently, the lack of a robust phylogenetic framework for the Klebsormidiophyceae hampers our understanding of the evolutionary history of these key traits. Here, we conducted a phylogenomic analysis utilizing advanced models that can counteract systematic biases. We sequenced 24 new transcriptomes of Klebsormidiophyceae and combined them with 14 previously published genomic and transcriptomic datasets. Using an analysis built on 845 loci and sophisticated mixture models, we establish a phylogenomic framework, dividing the six distinct genera of Klebsormidiophyceae in a novel three-order system, with a deep divergence more than 830 million years ago. Our reconstructions of ancestral states suggest (1) an evolutionary history of multiple transitions between terrestrial-aquatic habitats, with stem Klebsormidiales having conquered land earlier than embryophytes, and (2) that the body plan of the last common ancestor of Klebsormidiophyceae was multicellular, with a high probability that it was filamentous whereas the sarcinoids and unicells in Klebsormidiophyceae are likely derived states. We provide evidence that the first multicellular streptophytes likely lived about a billion years ago.

Comparative analysis of SPL transcription factors from streptophyte algae and embryophytes reveals evolutionary trajectories of SPL family in streptophytes.

SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) genes encode plant-specific transcription factors which are important regulators of diverse plant developmental processes. We took advantage of available genome sequences of streptophyte algae representatives to investigate the relationships of SPL genes between freshwater green algae and land plants. Our analysis showed that streptophyte algae, hornwort and liverwort genomes encode from one to four SPL genes which is the smallest set, in comparison to other land plants studied to date. Based on the phylogenetic analysis, four major SPL phylogenetic groups were distinguished with Group 3 and 4 being sister to Group 1 and 2. Comparative motif analysis revealed conserved protein motifs within each phylogenetic group and unique bryophyte-specific motifs within Group 1 which suggests lineage-specific protein speciation processes. Moreover, the gene structure analysis also indicated the specificity of each by identifying differences in exon-intron structures between the phylogenetic groups, suggesting their evolutionary divergence. Since current understanding of SPL genes mostly arises from seed plants, the presented comparative and phylogenetic analyzes from freshwater green algae and land plants provide new insights on the evolutionary trajectories of the SPL gene family in different classes of streptophytes.

A mysterious cloak: the peptidoglycan layer of algal and plant plastids.

The plastids of algae and plants originated on a single occasion from an endosymbiotic cyanobacterium at least a billion years ago. Despite the divergent evolution that characterizes the plastids of different lineages, many traits such as membrane organization and means of fission are universal-they pay tribute to the cyanobacterial origin of the organelle. For one such trait, the peptidoglycan (PG) layer, the situation is more complicated. Our view on its distribution keeps on changing and little is known regarding its molecular relevance, especially for land plants. Here, we investigate the extent of PG presence across the Chloroplastida using a phylogenomic approach. Our data support the view of a PG layer being present in the last common ancestor of land plants and its remarkable conservation across bryophytes that are otherwise characterized by gene loss. In embryophytes, the occurrence of the PG layer biosynthetic toolkit becomes patchier and the availability of novel genome data questions previous predictions regarding a functional coevolution of the PG layer and the plastid division machinery-associated gene FtsZ3. Furthermore, our data confirm the presence of penicillin-binding protein (PBP) orthologs in seed plants, which were previously thought to be absent from this clade. The 5-7 nm thick, and seemingly unchanged, PG layer armoring the plastids of glaucophyte algae might still provide the original function of structural support, but the same can likely not be said about the only recently identified PG layer of bryophyte and tracheophyte plastids. There are several issues to be explored regarding the composition, exact function, and biosynthesis of the PG layer in land plants. These issues arise from the fact that land plants seemingly lack certain genes that are believed to be crucial for PG layer production, even though they probably synthesize a PG layer.

Hidden functional complexity in the flora of an early land ecosystem.

The first land ecosystems were composed of organisms considered simple in nature, yet the morphological diversity of their flora was extraordinary. The biological significance of this diversity remains a mystery largely due to the absence of feasible study approaches. To study the functional biology of Early Devonian flora, we have reconstructed extinct plants from fossilised remains in silico. We explored the morphological diversity of sporangia in relation to their mechanical properties using finite element method. Our approach highlights the impact of sporangia morphology on spore dispersal and adaptation. We discovered previously unidentified innovations among early land plants, discussing how different species might have opted for different spore dispersal strategies. We present examples of convergent evolution for turgor pressure resistance, achieved by homogenisation of stress in spherical sporangia and by torquing force in Tortilicaulis-like specimens. In addition, we show a potential mechanism for stress-assisted sporangium rupture. Our study reveals the deceptive complexity of this seemingly simple group of organisms. We leveraged the quantitative nature of our approach and constructed a fitness landscape to understand the different ecological niches present in the Early Devonian Welsh Borderland flora. By connecting morphology to functional biology, these findings facilitate a deeper understanding of the diversity of early land plants and their place within their ecosystem.

Evolution of immunity networks across embryophytes.

Land plants (embryophytes), including vascular (tracheophytes) and non-vascular plants (bryophytes), co-evolved with microorganisms since descendants of an algal ancestor colonized terrestrial habitats around 500 million years ago. To cope with microbial pathogen infections, embryophytes evolved a complex immune system for pathogen perception and activation of defenses. With the growing number of sequenced genomes and transcriptome datasets from algae, bryophytes, tracheophytes, and available plant models, comparative analyses are increasing our understanding of the evolution of molecular mechanisms underpinning immune responses in different plant lineages. In this review, recent progress on plant immunity networks is highlighted with emphasis on the identification of key components that shaped immunity against pathogens in bryophytes compared to angiosperms during plant evolution.