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The agricultural sugarcane residues, bagasse and straws, can be used for second-generation ethanol (2GE) production by the cellulose conversion into glucose (saccharification). However, the lignin content negatively impacts the saccharification process. This polymer is mainly composed of guaiacyl (G), hydroxyphenyl (H), and syringyl (S) units, the latter formed in the ferulate 5-hydroxylase (F5H) branch of the lignin biosynthesis pathway. We have generated transgenic lines overexpressing ShF5H1 under the control of the C4H (cinnamate 4-hydroxylase) rice promoter, which led to a significant increase of up to 160% in the S/G ratio and 63% in the saccharification efficiency in leaves. Nevertheless, the content of lignin was unchanged in this organ. In culms, neither the S/G ratio nor sucrose accumulation was altered, suggesting that ShF5H1 overexpression would not affect first-generation ethanol production. Interestingly, the bagasse showed a significantly higher fiber content. Our results indicate that the tissue-specific manipulation of the biosynthetic branch leading to S unit formation is industrially advantageous and has established a foundation for further studies aiming at refining lignin modifications. Thus, the ShF5H1 overexpression in sugarcane emerges as an efficient strategy to improve 2GE production from straw.

n the Neotropics, the focus of apomictic studies predominantly centres on trees within the Brazilian savanna, characterized, mostly as sporophytic and facultative, associated with polyploidy and polyembryony. To enhance our understanding of the mechanisms governing apomixis and sexual reproduction in tropical herbaceous plants, we clarify the relationship between apomixis, chromosome counts, and polyembryony in the epiphytic orchid Zygopetalum mackayi , which forms a polyploid complex within rocky outcrops in both the Brazilian savanna and the Atlantic forest. To define embryo origins and describe megasporogenesis and megagametogenesis, we performed manual self-pollinations in first-day flowers of cultivated plants, considering all three cytotypes (2 x , 3 x , 4 x ) of this species. Flowers and fruits at different stages were collected to describe the development and morphology of ovules and seeds considering sexual and apomictic processes. As self-pollination treatments resulted in high fruit abortion in diploids, we also examined pollen tube development in aborted flowers and fruits to search for putative anomalies. Megasporogenesis and megagametogenesis occur regularly in all cytotypes. Apomixis is facultative and sporophytic, and associated with polyploid cytotypes, while diploid individuals exclusively engage in sexual reproduction. Polyembryony is caused mainly by the production of adventitious embryos from nucellar cells of triploids and tetraploids, but also by the development of multiple archesporia in all cytotypes. Like other apomictic angiosperms within the Brazilian savanna, our findings demonstrate that apomixis in Z. mackayi relies on pollinators for seed production. We also consider the ecological implications of these apomictic patterns in Z. mackayi within the context of habitat loss and its dependence on pollinators., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Annals of Botany Company.)

There is an increasing need for renewable energy sources to replace part of our fossil fuel-based economy and reduce greenhouse gas emission. Sugarcane bagasse is a prominent feedstock to produce cellulosic bioethanol, but strategies are still needed to improve the cost-effective exploitation of this potential energy source. In model plants, it has been shown that GUX genes are involved in cell wall hemicellulose decoration, adding glucuronic acid substitutions on the xylan backbone. Mutation of GUX genes increases enzyme access to cell wall polysaccharides, reducing biomass recalcitrance in Arabidopsis thaliana. Here, we characterized the sugarcane GUX genes and silenced GUX2 in commercial hybrid sugarcane. The transgenic lines had no penalty in development under greenhouse conditions. The sugarcane GUX1 and GUX2 enzymes generated different patterns of xylan glucuronidation, suggesting they may differently influence the molecular interaction of xylan with cellulose and lignin. Studies using biomass without chemical or steam pretreatment showed that the cell wall polysaccharides, particularly xylan, were less recalcitrant in sugarcane with GUX2 silenced than in WT plants. Our findings suggest that manipulation of GUX in sugarcane can reduce the costs of second-generation ethanol production and enhance the contribution of biofuels to lowering the emission of greenhouse gases., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Premise: Pogoniopsis likely represents an independent photosynthesis loss in orchids. We use phylogenomic data to better identify the phylogenetic placement of this fully mycoheterotrophic taxon, and investigate its molecular evolution., Methods: We performed likelihood analysis of plastid and mitochondrial phylogenomic data to localize the position of Pogoniopsis schenckii in orchid phylogeny, and investigated the evolution of its plastid genome., Results: All analyses place Pogoniopsis in subfamily Epidendroideae, with strongest support from mitochondrial data, which also place it near tribe Sobralieae with moderately strong support. Extreme rate elevation in Pogoniopsis plastid genes broadly depresses branch support; in contrast, mitochondrial genes are only mildly rate elevated and display very modest and localized reductions in bootstrap support. Despite considerable genome reduction, including loss of photosynthesis genes and multiple translation apparatus genes, gene order in Pogoniopsis plastomes is identical to related autotrophs, apart from moderately shifted inverted repeat (IR) boundaries. All cis-spliced introns have been lost in retained genes. Two plastid genes (accD, rpl2) show significant strengthening of purifying selection. A retained plastid tRNA gene (trnE-UUC) of Pogoniopsis lacks an anticodon; we predict that it no longer functions in translation but retains a secondary role in heme biosynthesis., Conclusions: Slowly evolving mitochondrial genes clarify the placement of Pogoniopsis in orchid phylogeny, a strong contrast with analysis of rate-elevated plastome data. We documented the effects of the novel loss of photosynthesis: for example, despite massive gene loss, its plastome is fully colinear with other orchids, and it displays only moderate shifts in selective pressure in retained genes., (© 2022 Botanical Society of America.)

Fully mycoheterotrophic plants can be difficult to place in plant phylogeny due to elevated substitution rates associated with photosynthesis loss. This potentially limits the effectiveness of downstream analyses of mycoheterotrophy that depend on accurate phylogenetic inference. Although mitochondrial genomic data sets are rarely used in plant phylogenetics, theory predicts that they should be resilient to long-branch artefacts, thanks to their generally slow evolution, coupled with limited rate elevation in heterotrophs. We examined the utility of mitochondrial genomes for resolving contentious higher-order placements of mycoheterotrophic lineages in two test cases: monocots (focusing on Dioscoreales) and Ericaceae. We find Thismiaceae to be distantly related to Burmanniaceae in the monocot order Dioscoreales, conflicting with current classification schemes based on few gene data sets. We confirm that the unusual Afrothismia is related to Taccaceae-Thismiaceae, with a corresponding independent loss of photosynthesis. In Ericaceae we recovered the first well supported relationships among its five major lineages: mycoheterotrophic Ericaceae are not monophyletic, as pyroloids are inferred to be sister to core Ericaceae, and monotropoids to arbutoids. Genes recovered from mitochondrial genomes collectively resolved previously ambiguous mycoheterotroph higher-order relationships. We propose that mitochondrial genomic data should be considered in standardised gene panels for inferring overall plant phylogeny., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Premise: Unlike most flowering plants, orchid flowers have under-developed ovules that complete development only after pollination. Classical studies reported variation in the stage in which ovule development is arrested, but the extent of this variation and its evolutionary and ecological significance are unclear., Methods: Here, we used light microscopy to observe ovule development at anthesis for 39 species not previously studied and surveyed the literature gaining information on 94 orchid species. Tropical and temperate members of all five orchid subfamilies as well as species with contrasting pollination strategies (rewarding versus deceptive) and life forms (epiphytic versus terrestrial) were represented. We analyzed the data using statistical comparisons and a phylogenetic generalized least square (PGLS) analysis., Results: Apostasioideae, the sister to the rest of the orchids, have mature ovules similar to other Asparagales, while under-differentiated ovules are present in the other subfamilies. Ovule developmental stages showed high variation even among closely related groups. Ovules were more developed in terrestrial than in epiphytic, in temperate than in tropical, and in rewarding than in deceptive pollination orchid species. This latter comparison was also significant in the PGLS analysis., Conclusions: These results suggest that ovule developmental stage in orchids can be shaped by ecological factors, such as seasonality and pollination strategy, and can be selected for optimizing female reproductive investment., (© 2021 Botanical Society of America.)

Background: Pogoniopsis schenckii Cogn. is a mycoheterotrophic orchid that can be used as a model to understand the influence of mycoheterotrophy at different stages of the reproductive cycle. We aimed to verify the presence of endophytic and epiphytic fungi at each stage of the reproductive process and investigated how the breeding system may relate to genetic structure and diversity of populations. In this study we performed anatomical and ultrastructural analyses of the reproductive organs, field tests to confirm the breeding system, and molecular analysis to assess genetic diversity and structure of populations., Results: During the development of the pollen grain, embryo sac and embryogenesis, no fungal infestation was observed. The presence of endophytic fungal hyphae was observed just within floral stems and indehiscent fruit. Beyond assuring the presence of fungus that promote seed germination, specific fungi hyphae in the fruit may affect other process, such as fruit ripening. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations., Conclusions: We discuss an interesting interaction: fungal hyphae in the indehiscent fruit. These fungal hyphae seem to play different roles inside fruit tissues, such as acting in the fruit maturation process and increasing the proximity between fungi and plant seeds even before dispersion occurs. As other mycoheterotrophic orchids, P. schenckii is autogamous, which may explain the low genetic diversity and high genetic structure in populations. Altogether, our findings provide important novel information about the mechanisms shaping ecology and evolution of fragmented populations of mycoheterotrophic plant., (© 2021. The Author(s).)

Lignin is a heterogeneous aromatic polymer responsible for cell wall stiffness and protection from pathogen attack. However, lignin represents a bottleneck to biomass degradation due to its recalcitrance related to the natural cell wall resistance to release sugars for fermentation or further processing. A biological approach involving genetics and molecular biology was used to disrupt lignin pathway synthesis and decrease lignin deposition. Here, we imaged three-dimensional fragments of the petioles of wild type and C4H lignin mutant Arabidopsis thaliana plants by synchrotron cryo-ptychography. The three-dimensional images revealed the heterogeneity of vessels, parenchyma, and fibre cell wall morphologies, highlighting the relation between disturbed lignin deposition and vessel implosion (cell collapsing and obstruction of water flow). We introduce a new parameter to accurately define cell implosion conditions in plants, and we demonstrate how cryo-ptychographic X-ray computed tomography (cryo-PXCT) provides new insights for plant imaging in three dimensions to understand physiological processes.

Endophytic fungi are those that inhabit within organs and tissues without causing damage, while mycorrhizal fungi develop hyphal complexes called pelotons within cortical cells of orchid roots. Although abundant and frequent in all plant organs, the role of endophytic fungi has been neglected in relation to orchid's early development. Pogoniopsis schenckii Cogn. is an aclorophyllated and mycoheterotrophic (MH) orchid. This study aimed at i) investigating the endophytic fungal community in organs of P. schenckii and its mycorrhizal fungi associated; ii) evaluating the ability of isolated fungus in the in vitro germination of the seeds of the species, and iii) describing the development of P. schenckii protocorm, analyzing the ultrastructure of the infected cells. Six genera of fungi were isolated and identified through the partial sequencing of the internal transcribed spacer region, all belonging to the phylum Ascomycota. Also, Tulasnellaceae was identified through uncultured technique as potentially mycorrhizal in this MH orchid. Some isolates of the genera Trichoderma , Fusarium , and especially Clonostachys presented germinative potential on P. schenckii seeds, causing rupture of the external tegument. The protocorms showed complete absence of peloton formation, but fungal hyphae were clearly observed within living cells. This is the first report of germination of a MH and aclorophyllated orchid species stimulated by the presence of non-mycorrhizal endophytic fungi isolated from fruits and roots of the same species., (Copyright © 2019 Sisti, Flores-Borges, Andrade, Koehler, Bonatelli and Mayer.)

The number of integuments found in angiosperm ovules is variable. In orchids, most species show bitegmic ovules, except for some mycoheterotrophic species that show ovules with only one integument. Analysis of ovules and the development of the seed coat provide important information regarding functional aspects such as dispersal and seed germination. This study aimed to analyze the origin and development of the seed coat of the mycoheterotrophic orchid Pogoniopsis schenckii and to compare this development with that of other photosynthetic species of the family. Flowers and fruits at different stages of development were collected, and the usual methodology for performing anatomical studies, scanning microscopy, and transmission microscopy following established protocols. P. schenckii have ategmic ovules, while the other species are bitegmic. No evidence of integument formation at any stage of development was found through anatomical studies. The reduction of integuments found in the ovules could facilitate fertilization in this species. The seeds of P. schenckii , Vanilla planifolia , and V. palmarum have hard seed coats, while the other species have seed coats formed by the testa alone, making them thin and transparent. P. schenckii, in contrast to the other species analyzed, has a seed coat that originates from the nucellar epidermis, while in other species, the seed coat originates from the outer integument., (Copyright © 2019 Alves, Pinheiro, Niedzwiedzki and Mayer.)

Coffea arabica is an allotetraploid of high economic importance. C. arabica transcriptome is a combination of the transcripts of two parental genomes (C. eugenioides and C. canephora) that gave rise to the homeologous genes of the species. Previous studies have reported the transcriptional dynamics of C. arabica. In these reports, the ancestry of homeologous genes was identified and the overall regulation of homeologous differential expression (HDE) was explored. One of these genes is part of the FRIGIDA-like family (FRL), which includes the Arabidopsis thaliana flowering-time regulation protein, FRIGIDA (FRI). As nonfunctional FRI proteins give rise to rapid-cycling summer annual ecotypes instead of vernalization-responsive winter-annuals, allelic variation in FRI can modulate flowering time in A. thaliana. Using bioinformatics, genomic analysis, and the evaluation of gene expression of homeologs, we characterized the FRL gene family in C. arabica. Our findings indicate that C. arabica expresses 10 FRL homeologs, and that, throughout flower and fruit development, these genes are differentially transcribed. Strikingly, in addition to confirming the expression of FRL genes during zygotic embryogenesis, we detected FRL expression during direct somatic embryogenesis, a novel finding regarding the FRL gene family. The HDE profile of FRL genes suggests an intertwined homeologous gene regulation. Furthermore, we observed that FLC gene of C. arabica has an expression profile similar to that of CaFRL genes.

Colleters are trichomes or emergencies that produce a sticky exudate consisting of a mixture of mucilage, lipids, terpenes, and phenolic compounds. Colleters occur in at least 60 families of angiosperms; however, reports of them are scarce for the Orchidaceae. Elleanthus brasiliensis is distinguished by the presence of an abundant gelatinous secretion that covers almost all of its inflorescences. We aimed to describe the histology of colleters in inflorescences of E. brasiliensis and Elleanthus crinipes , and to analyze the chemical composition of their secretion to better understand the functions of these secretory structures. Due to the low frequency of colleters and lack of visible secretion in E. crinipes , histochemical tests and chemical analyses were not performed for this species. Colleters are of a brush type and their secretion has, at the same time, hydrophilic and lipophilic components. Histochemical tests further revealed the presence of pectin, mucilage, lipids, terpenes, phenolic compounds, and proteins. The GC-MS analysis confirmed the presence of γ-sitosterol and palmitic, linoleic, and stearic acids in the secretion of E. brasiliensis . Infrared analysis indicated the possible presence of polysaccharides in the secretion. The occurrence of colleters in both species studied and in other orchids described in the literature suggests that these structures are common in the inflorescences of tropical orchids. In these environments, the hydrated polysaccharides in the secretion form a dense matrix that can act as a physical barrier, and terpenes may help to protect against herbivores and pathogenic microorganisms. This information broadens our knowledge of the morphological and chemical diversity of the secretions produced by orchid colleters.

We used primers designed on conserved gene regions of several species to isolate the most expressed genes of the lignin pathway in four Saccharum species. S. officinarum and S. barberi have more sucrose in the culms than S. spontaneum and S. robustum, but less polysaccharides and lignin in the cell wall. S. spontaneum, and S. robustum had the lowest S/G ratio and a lower rate of saccharification in mature internodes. Surprisingly, except for CAD, 4CL, and CCoAOMT for which we found three, two, and two genes, respectively, only one gene was found for the other enzymes and their sequences were highly similar among the species. S. spontaneum had the highest expression for most genes. CCR and CCoAOMT B presented the highest expression; 4CL and F5H showed increased expression in mature tissues; C3H and CCR had higher expression in S. spontaneum, and one of the CADs isolated (CAD B) had higher expression in S. officinarum. The similarity among the most expressed genes isolated from these species was unexpected and indicated that lignin biosynthesis is conserved in Saccharum including commercial varieties Thus the lignin biosynthesis control in sugarcane may be only fully understood with the knowledge of the promotor region of each gene.

In the xylem of angiosperm plants, microscopic pits through the secondary cell walls connect the water-conducting vessels. Cellulosic meshes originated from primary walls, and middle lamella between adjacent vessels, called the pit membrane, separates one conduit from another. The intricate structure of the nano-sized pores in pit membranes enables the passage of water under negative pressure without hydraulic failure due to obstruction by gas bubbles (i.e. embolism) under normal conditions or mild drought stress. Since the chemical composition of pit membranes affects embolism formation and bubble behavior, we directly measured pit membrane composition in Populus nigra wood. Here, we characterized the chemical composition of cell wall structures by synchrotron infrared nanospectroscopy and atomic force microscopy-infrared nanospectroscopy with high spatial resolution. Characteristic peaks of cellulose, phenolic compounds, and proteins were found in the intervessel pit membranes of P. nigra wood. In addition, the vessel to parenchyma pit membranes and developing cell walls of the vascular cambium showed clear signals of cellulose, proteins, and pectin. We did not find a distinct peak of lignin and other compounds in these structures. Our investigation of the complex chemical composition of intervessel pit membranes furthers our understanding of the flow of water and bubbles between neighboring conduits. The advances presented here pave the way for further label-free studies related to the nanochemistry of plant cell components., (© 2018 American Society of Plant Biologists. All rights reserved.)

Eithea lagopaivae Campos-Rocha & Dutilh, sp. nov. is described as the second species of the formerly monotypic genus Eithea . It is characterized by a one flowered inflorescence, completely hollow scape, white or lightly magenta-striated flower that is enclosed by spathe bracts fused for more than the lower fifth of its length. Comments on its range, habitat, phenology, as well as photographs and illustrations are provided. In addition, a distribution map and an identification key for the two species of the genus are presented and anatomical and ecological differences compared. Known by only two small populations exposed to several types of threats and without any guarantee of protection, E. lagopaivae is considered a Critically Endangered (CR) species.