TgLUT1 regulated by TgWRKY10 enhances the tolerance of Torreya grandis to drought stress.

Drought stress is a major abiotic stress which severely reduces the plant growth and limits agricultural productivity. Previous studies have demonstrated that lutein directly synthesized by the carotenoid epsilon-ring hydroxylase gene (LUT1) played crucial roles in regulating drought response. Notwithstanding the myriad studies on LUT1's response to drought stress in certain plant species such as Arabidopsis, the precise function mechanisms within tree species remain ambiguously understood. Our study reveals that under drought stress, TgLUT1, a novel LUT gene instrumental in ß-lutein biosynthesis, was markedly up-regulated in Torreya grandis. Subcellular localization assay indicated that TgLUT1 protein was localized to chloroplasts. Phenotypic analysis showed that overexpression of TgLUT1 enhanced the tolerance of tomato to drought stress. Overexpressing of TgLUT1 increased the values of maximal photochemical efficiency of photosystem II (Fv/Fm), net photosynthetic rate (Pn) and non-photochemical quenching (NPQ), and reduced the accumulation of hydrogen peroxide (H2O2), malondialdehyde (MDA) content and electrolyte leakage percentage in response to drought stress. Furthermore, overexpression of TgLUT1 decreased the stomatal conductance to reduce the water loss rate exposed to drought stress. In addition, yeast one-hybrid assay, dual luciferase assay system and qRT-PCR results showed that TgWRKY10 down-regulated by drought stress inhibited the expression of TgLUT1 by directly binding to the TgLUT1 promoter. Collectively, our results show that TgWRKY10, down-regulated by drought stress, negatively regulates the expression of TgLUT1 to modulate the drought stress response. This study contributes to a more comprehensive understanding of LUT1's function in the stress responses of economically significant forest plants.

Integrated Metabolomics, Transcriptome and Functional Analysis Reveal Key Genes Are Involved in Tree Age-Induced Amino Acid Accumulation in Torreya grandis Nuts.

Torreya grandis is native Chinese tree species of economic significance, renowned for its long lifespan and the rich nutritional value of its nuts. In this study, we analyzed the morphological characteristics, metabolites, associated gene expressions, and regulatory mechanism in nuts from young (10 years old) and old (1000 years old) T. grandis trees. We observed that the length, width, and weight of nuts from older trees were considerably greater than those from younger trees. Metabolomic analysis revealed that the concentrations of 18 amino acids and derivatives (including histidine and serine) in nuts from older trees were markedly higher than those in nuts from younger trees. Transcriptome and metabolomic correlation analysis identified 16 genes, including TgPK (pyruvate kinase), TgGAPDH (glyceraldehyde 3-phosphate dehydrogenase), and others, which exhibit higher expression levels in older trees compared to younger trees, as confirmed by qRT-PCR. These genes are associated with the biosynthesis of histidine, glutamic acid, tryptophan, and serine. Transient expression of TgPK in tobacco led to increased pyruvate kinase activity and amino acid content (histidine, tryptophan, and serine). Additionally, dual-luciferase assays and yeast one-hybrid results demonstrated that TgWRKY21 positively regulates TgPK expression by directly binding to the TgPK promoter. These findings not only demonstrate the nutritional differences between nuts from young and old trees but also offer fresh insights into the development of nutritional sources and functional components based on nuts from old trees, enriching our understanding of the potential benefits of utilizing nuts from older trees.

TgLCYB1 regulated by TgWRKY22 enhances the tolerance of Torreya grandis to waterlogging stress.

ß-Carotene functions in plant growth and development and plays an important role in resisting abiotic stress, such as drought and salt stress. The specific function and mechanism by which ß-carotene responds to waterlogging stress, however, remain elusive. In this study, we found that ß-carotene content and lycopene cyclase (TgLCYB1) expression, both in leaves and roots of Torreya grandis, were increased under waterlogging treatment. Subcellular localization assays indicated that TgLCYB1 was localized in the chloroplasts. Phenotypic, physiological, and metabolome analysis showed that overexpression of TgLCYB1 enhanced the tolerance of tomato plants to waterlogging stress. Furthermore, application of a LCYB enzyme inhibitor, 2-(4-chlorophenylthio)-triethylamine hydrochloride, markedly enhanced the sensitivity of T. grandis to waterlogging stress. In addition, yeast one-hybrid assay, the dual luciferase assay system, and real-time quantitative PCR indicated that waterlogging stress induced TgWRKY22 to increase TgLCYB1 expression by binding to the TgLCYB1 promoter. Collectively, our results indicated that TgWRKY22 positively regulated TgLCYB1 expression to improve the activities of antioxidant enzyme and increase the levels of some key metabolites, thereby relieving waterlogging-induced oxidative damage, and consequently modulating the waterlogging stress response. This study contributes to a more comprehensive understanding of carotenoid functions and the role LCYB genes play in plant stress response.

Transcription factors TgbHLH95 and TgbZIP44 cotarget terpene biosynthesis gene TgGPPS in Torreya grandis nuts.

Terpenes are volatile compounds responsible for aroma and the postharvest quality of commercially important xiangfei (Torreya grandis) nuts, and there is interest in understanding the regulation of their biosynthesis. Here, a transcriptomics analysis of xiangfei nuts after harvest identified 156 genes associated with the terpenoid metabolic pathway. A geranyl diphosphate (GPP) synthase (TgGPPS) involved in production of the monoterpene precursor GPP was targeted for functional characterization, and its transcript levels positively correlated with terpene levels. Furthermore, transient overexpression of TgGPPS in tobacco (Nicotiana tabacum) leaves or tomato (Solanum lycopersicum) fruit led to monoterpene accumulation. Analysis of differentially expressed transcription factors identified one basic helix-loop-helix protein (TgbHLH95) and one basic leucine zipper protein (TgbZIP44) as potential TgGPPS regulators. TgbHLH95 showed significant transactivation of the TgGPPS promoter, and its transient overexpression in tobacco leaves led to monoterpene accumulation, whereas TgbZIP44 directly bound to an ACGT-containing element in the TgGPPS promoter, as determined by yeast 1-hybrid test and electrophoretic mobility shift assay. Bimolecular fluorescence complementation, firefly luciferase complementation imaging, co-immunoprecipitation, and GST pull-down assays confirmed a direct protein-protein interaction between TgbHLH95 and TgbZIP44 in vivo and in vitro, and in combination these proteins induced the TgGPPS promoter up to 4.7-fold in transactivation assays. These results indicate that a TgbHLH95/TgbZIP44 complex activates the TgGPPS promoter and upregulates terpene biosynthesis in xiangfei nuts after harvest, thereby contributing to its aroma.

Metabolomics reveal changes in flavor quality and bioactive components in post-ripening Torreya grandis nuts and the underlying mechanism.

Secondary metabolites are a group of small molecules with critical roles in plants fitness in addition to their potential bioactivities in humans. Most of these compounds are associated with the flavor and quality formation of fruits or nuts during the development or the postharvest stages. Change in metabolic profiles and shifts underpinning the post-ripening process in T. grandis nuts are not yet reported. In this study, a large scale untargeted metabolomics approach was employed in T. grandis nuts, revealing for a total of 140 differential accumulated metabolites. Among them, nearly 60% of metabolites belonging to terpenoids, coumarins and phenolic acids, and phytohormones were showed a gradual accumulation pattern, while most of compounds in flavonoids were decreased during post-ripening. An in-depth analysis of changes in these metabolite classes suggest a framework for post-ripening process effect associated with the postharvest quality of T. grandis nuts for the first time.

Ethylene treatment promotes umami taste-active amino acids accumulation of Torreya grandis nuts post-harvest by comparative chemical and transcript analyses.

Amino acids play critical roles in physiological processes and also contribute significantly to fruit quality. In this study, the effect of exogenous ethylene on amino acids metabolism and related genes expression in Torreya grandis were investigated. The results revealed that ethylene treatment (3000 µL L-1 for 24 h) significantly increased amino acids level. Umami amino acids were distinctly upregulated in ethylene-treated versus control nuts, with glutamic and aspartic acids to demonstrate 1.9-fold and 2.1-fold increase. Transcriptome analysis revealed that deferentially expressed genes were mainly enriched in alanine aspartate and glutamate metabolism. RT-qPCR confirmed that ethylene treatment up-regulated expression of their biosynthesis genes (TgGOGAT1, TgAATC1, TgAATC4) concurrent with suppression of their degradation enzymes (TgGS2, TgGAD1, TgGAD3, TgASNS1). Ethylene treatment appears to promote umami taste-active amino acids and improve T. grandis nut quality post-harvest.

A comprehensive metabolomics analysis of Torreya grandis nuts with the effective de-astringent treatment during the postharvest ripening stage.

Astringency removal is important for the quality of Torreya grandis nut and occurs after harvest. Here, we evaluated the effect of NaHCO3 treatment on astringency removal and compared the differential metabolites of the seed coat and kernel using a UHPLC QQQ-MS-based metabolomics approach. The result revealed the nut astringency was primarily enriched in the seed coat with more soluble tannins. The NaHCO3 treatment greatly shortened the de-astringency process, as indicated by a faster conversion of soluble tannins to insoluble tannins and more acetaldehyde production. Besides, a total of 293 metabolites, including 92 phenolic acids and 37 flavonoids, were tentatively characterized in the seed coat. A further comparative analysis of the metabolomics indicated epigallocatechin, gallocatechin, catechin, procyanidin B1, B2, B3 and C1 to be the major metabolites influenced by the NaHCO3 treatment. This study provides new insights regarding the metabolite differences of Torreya grandis nuts processed with different de-astringent treatments.

Unraveling the malate biosynthesis during development of Torreya grandis nuts.

Torreya grandis is a characteristic rare economic tree species in subtropical mountainous areas. The kernels of T. grandis have rich content of organic acids, and malate is the predominant organic acid in T. grandis kernels. However, the contents, biosynthesis/metabolism pathway and transcriptional regulation of malate in developing T. grandis kernels remain completely unknown. Here, the organic acid composition in developing T. grandis kernels was first analyzed. The results showed that the content of malate was increased during the maturation T. grandis kernels. A malate synthase (TgMLS) gene might be involved in the accumulation of malate based on transcriptome data, gene expression and enzyme activity analysis. Transient expression of TgMLS in tobacco resulted in the high malate synthase activity and malate content. Furthermore, a basic helix-loop-helix transcription factor (bHLH), TgbHLH87 was identified to positively regulate the TgMLS expression via directly binding the TgMLS promoter. Our finding contributes to mechanism underlying malate accumulation in T. grandis kernels.

New insights into the accumulation of vitamin B3 in Torreya grandis nuts via ethylene induced key gene expression.

Vitamin B3, derived primarily from plant sources, is an essential nutrient for humans. Torreya grandis is rich in vitamin B3, however, the mechanism underlying the biosynthesis and regulation of vitamin B3 in T. grandis remains unclear. A systematic transcriptomic investigation was thus conducted to identify the gene expression pattern of vitamin B3 biosynthesis in 10 T. grandis cultivars. The findings suggest that biosynthesis occurs mainly via the aspartate pathway. Expression and correlation analyses indicate that aspartate oxidase (AOX) and quinolinate synthase (QS) may play important roles in vitamin B3 accumulation. Furthermore, co-expression network and ethephon treatments indicate that the ethylene response factor (ERF) may be involved in the regulation of vitamin B3 biosynthesis in T. grandis nuts. Our findings not only help to elucidate the biosynthesis of vitamin B3, but also provide valuable resource material for future genomic research and molecular-assisted breeding to develop genotypes with higher vitamin B3 levels.

The interaction of temperature and relative humidity affects the main aromatic components in postharvest Torreya grandis nuts.

The postharvest ripening stage is necessary for Torreya grandis (T. grandis) nuts to complete aromatic synthesis, which requires appropriate temperature and relative humidity (RH). Currently, scarce information is available regarding the changes in aroma profiles in T. grandis nuts and the relationship with their response to different environmental conditions. Therefore, the interaction of temperature (20 °C or 30 °C) and relative humidity (70% RH or 90% RH) was investigated on aromatic substances after harvest. The results showed that 56 aromatic components were detected by a gas chromatography-mass spectrometer (GC-MS) and mainly divided into five categories, among which terpenes were the most abundant (56.2-86.7%). Principal component analysis (PCA) showed that both temperature and humidity can affect the aroma composition, and terpenes were mainly influenced by humidity. Specifically, d-limonene occupied the largest proportion of terpenes (63.0-90.8%) and was significantly upregulated by high humidity.

The effect of ethylene on squalene and ß-sitosterol biosynthesis and its key gene network analysis in Torreya grandis nuts during post-ripening process.

Squalene and ß-sitosterol are health-benefit compounds due to their nutritional and medicinal properties. It has been reported that the content of these bioactive compounds is relatively high in Torreya grandis nuts. However, it is not yet known what changes in squalene and ß-sitosterol accumulation occur during the special post-ripening process of T. grandis nuts and the effect of the well-known ripening hormone ethylene on the regulatory mechanism of their biosynthetic pathways. Thus, we performed transcriptome and metabolite analyses. The results showed that ethylene not only promoted the post-ripening process but also enhanced the accumulation of squalene by inducing gene expression in the mevalonate pathway. At the same time, ethylene treatment also promoted the accumulation of other sterols but inhibited gene expression in the ß-sitosterol biosynthesis pathway. In addition, co-expression and correlation analysis suggested a framework for the transcriptional regulation of squalene and ß-sitosterol biosynthesis genes under ethylene treatment.

Acceleration of Aril Cracking by Ethylene in Torreya grandis During Nut Maturation.

Torreya grandis 'Merrillii' is a famous nut with great nutritional value and high medicinal value. Aril cracking is an important process for seed dispersal, which is also an indicator of seed maturation. However, the cracking mechanism of T. grandis aril during the maturation stage remains largely unknown. Here, we provided a comprehensive view of the physiological and molecular levels of aril cracking in T. grandis by systematically analyzing its anatomical structure, physiological parameters, and transcriptomic response during the cracking process. These results showed that the length of both epidermal and parenchymatous cell layers significantly increased from 133 to 144 days after seed protrusion (DASP), followed by a clear separation between parenchymatous cell layers and kernel, which was accompanied by a breakage between epidermal and parenchymatous cell layers. Moreover, analyses of cell wall composition showed that a significant degradation of cellular wall polysaccharides occurred during aril cracking. To examine the global gene expression changes in arils during the cracking process, the transcriptomes (96 and 141 DASP) were analyzed. KEGG pathway analysis of DEGs revealed that 4 of the top 10 enriched pathways were involved in cell wall modification and 2 pathways were related to ethylene biosynthesis and ethylene signal transduction. Furthermore, combining the analysis results of co-expression networks between different transcription factors, cell wall modification genes, and exogenous ethylene treatments suggested that the ethylene signal transcription factors (ERF11 and ERF1A) were involved in aril cracking of T. grandis by regulation of EXP and PME. Our findings provided new insights into the aril cracking trait in T. grandis.

Effect of Temperature and Humidity on Oil Quality of Harvested Torreya grandis cv. Merrillii Nuts During the After-Ripening Stage.

Temperature and relative humidity (RH) influence post-harvest ripening, a crucial stage for quality promotion in some oil plants or fruits. Torreya grandis cv. Merrillii nuts, which are rich in unsaturated fatty acids (UFA), are easily affected by temperature and humidity, and they oxidize quickly during the post-harvest ripening stage, leading to the deterioration of nut quality. In this study, the main nutraceutical components, fatty acid composition, and related metabolic parameters of lipid rancidity under four treatments (20°C and 70% RH, T20-LH; 30°C and 70% RH, T30-LH; 20°C and 90% RH, T20-HH; 30°C and 90% RH, T30-HH) were measured. The post-harvest ripening process was advanced under HH treatments (T20-HH and T30-HH) compared to LH treatments (T20-LH and T30-LH) and was associated with a shorter time for the seed coat to turn dark black and a faster reduction in starch content. The amount of unsaturated fatty acids significantly increased under the T20-HH treatment, but significantly decreased under the T30-HH treatment from 12 to 16 d of ripening time. The acid value (AV) and lipase activity under the T30-HH treatment remained virtually constant from 12 to 16 d of ripening time, and this was accompanied by a dramatic increase in peroxide value (POV), lipoxygenase (LOX) activity, and relative expression of the LOX2 gene. Meanwhile, a significant positive correlation between LOX activity and POV, malondialdehyde (MDA) content, and O2⋅- content was observed. The results imply that the lower amount of oxidative rancidity induced by the T20-HH treatment is related to the LOX activity induced by down-regulation of the LOX2 gene during the late after-ripening stage. Therefore, the T20-HH treatment not only promoted the post-harvest process of T. grandis 'Merrillii' nuts but also delayed lipid oxidation, which was ultimately associated with better oil quality at the late after-ripening stage.

Na2CO3-responsive Photosynthetic and ROS Scavenging Mechanisms in Chloroplasts of Alkaligrass Revealed by Phosphoproteomics.

Alkali-salinity exerts severe osmotic, ionic, and high-pH stresses to plants. To understand the alkali-salinity responsive mechanisms underlying photosynthetic modulation and reactive oxygen species (ROS) homeostasis, physiological and diverse quantitative proteomics analyses of alkaligrass (Puccinellia tenuiflora) under Na2CO3 stress were conducted. In addition, Western blot, real-time PCR, and transgenic techniques were applied to validate the proteomic results and test the functions of the Na2CO3-responsive proteins. A total of 104 and 102 Na2CO3-responsive proteins were identified in leaves and chloroplasts, respectively. In addition, 84 Na2CO3-responsive phosphoproteins were identified, including 56 new phosphorylation sites in 56 phosphoproteins from chloroplasts, which are crucial for the regulation of photosynthesis, ion transport, signal transduction, and energy homeostasis. A full-length PtFBA encoding an alkaligrass chloroplastic fructose-bisphosphate aldolase (FBA) was overexpressed in wild-type cells of cyanobacterium Synechocystis sp. Strain PCC 6803, leading to enhanced Na2CO3 tolerance. All these results indicate that thermal dissipation, state transition, cyclic electron transport, photorespiration, repair of photosystem (PS) II, PSI activity, and ROS homeostasis were altered in response to Na2CO3 stress, which help to improve our understanding of the Na2CO3-responsive mechanisms in halophytes.

New sights into lipid metabolism regulation by low temperature in harvested Torreya grandis nuts.

BACKGROUND: Torreya grandis, a large evergreen coniferous tree with oil-rich nuts, undergoes a crucial ripening stage after harvest that results in oil accumulation, finally giving rise to the nut that is edible in roasted form. To understand lipid metabolism in T. grandis nuts during the post-harvest ripening period, the effects of low temperature on lipid content, fatty acid composition, lipid biosynthesis and degradation were investigated. RESULTS: The lipid content increased during ripening at room temperature and a low temperature slowed down this increase. Linoleic acid content increased at low temperature, which was accompanied by an increase in the microsomal oleate desaturase (FAD2) activity and FAD2 expression. Furthermore, a low temperature attenuated lipid peroxidation as indicated by lower contents of malondialdehyde, hydroperoxide and total free fatty acid in T. grandis nuts during the ripening stage, as well as the down-regulation of gene expression of lipid degradation-related enzymes such as phospholipase D and lipoxygenases. CONCLUSION: The findings of the present study indicate that a low temperature increased polyunsaturated fatty acid contents by increasing FAD2 biosynthesis and decreasing lipid peroxidation, thereby improving the oil yield in T. grandis nuts during the post-harvest ripening period. © 2019 Society of Chemical Industry.

Full-Length Transcriptome Analysis of the Genes Involved in Tocopherol Biosynthesis in Torreya grandis.

The seeds of Torreya grandis (Cephalotaxaceae) are rich in tocopherols, which are essential components of the human diet as a result of their function in scavenging reactive oxygen and free radicals. Different T. grandis cultivars (10 cultivars selected in this study were researched, and their information is shown in Table S1 of the Supporting Information) vary enormously in their tocopherol contents (0.28-11.98 mg/100 g). However, little is known about the molecular basis and regulatory mechanisms of tocopherol biosynthesis in T. grandis kernels. Here, we applied single-molecule real-time (SMRT) sequencing to T. grandis (X08 cultivar) for the first time and obtained a total of 97 211 full-length transcripts. We proposed the biosynthetic pathway of tocopherol and identified eight full-length transcripts encoding enzymes potentially involved in tocopherol biosynthesis in T. grandis. The results of the correlation analysis between the tocopherol content and gene expression level in the 10 selected cultivars and different kernel developmental stages of the X08 cultivar suggested that homogentisate phytyltransferase coding gene ( TgVTE2b) and γ-tocopherol methyltransferase coding gene ( TgVTE4) may be key players in tocopherol accumulation in the kernels of T. grandis. Subcellular localization assays showed that both TgVTE2b and TgVTE4 were localized to the chloroplast. We also identified candidate regulatory genes similar to WRI1 and DGAT1 in Arabidopsis that may be involved in the regulation of tocopherol biosynthesis. Our findings provide valuable genetic information for T. grandis using full-length transcriptomic analysis, elucidating the candidate genes and key regulatory genes involved in tocopherol biosynthesis. This information will be critical for further molecular-assisted screening and breeding of T. grandis genotypes with high tocopherol contents.

Salinity Response in Chloroplasts: Insights from Gene Characterization.

Salinity is a severe abiotic stress limiting agricultural yield and productivity. Plants have evolved various strategies to cope with salt stress. Chloroplasts are important photosynthesis organelles, which are sensitive to salinity. An understanding of molecular mechanisms in chloroplast tolerance to salinity is of great importance for genetic modification and plant breeding. Previous studies have characterized more than 53 salt-responsive genes encoding important chloroplast-localized proteins, which imply multiple vital pathways in chloroplasts in response to salt stress, such as thylakoid membrane organization, the modulation of photosystem II (PS II) activity, carbon dioxide (CO2) assimilation, photorespiration, reactive oxygen species (ROS) scavenging, osmotic and ion homeostasis, abscisic acid (ABA) biosynthesis and signaling, and gene expression regulation, as well as protein synthesis and turnover. This review presents an overview of salt response in chloroplasts revealed by gene characterization efforts.

Na2CO3-responsive mechanisms in halophyte Puccinellia tenuiflora roots revealed by physiological and proteomic analyses.

Soil alkalization severely affects crop growth and agricultural productivity. Alkali salts impose ionic, osmotic, and high pH stresses on plants. The alkali tolerance molecular mechanism in roots from halophyte Puccinellia tenuiflora is still unclear. Here, the changes associated with Na2CO3 tolerance in P. tenuiflora roots were assessed using physiological and iTRAQ-based quantitative proteomic analyses. We set up the first protein dataset in P. tenuiflora roots containing 2,671 non-redundant proteins. Our results showed that Na2CO3 slightly inhibited root growth, caused ROS accumulation, cell membrane damage, and ion imbalance, as well as reduction of transport and protein synthesis/turnover. The Na2CO3-responsive patterns of 72 proteins highlighted specific signaling and metabolic pathways in roots. Ca(2+) signaling was activated to transmit alkali stress signals as inferred by the accumulation of calcium-binding proteins. Additionally, the activities of peroxidase and glutathione peroxidase, and the peroxiredoxin abundance were increased for ROS scavenging. Furthermore, ion toxicity was relieved through Na(+) influx restriction and compartmentalization, and osmotic homeostasis reestablishment due to glycine betaine accumulation. Importantly, two transcription factors were increased for regulating specific alkali-responsive gene expression. Carbohydrate metabolism-related enzymes were increased for providing energy and carbon skeletons for cellular metabolism. All these provide new insights into alkali-tolerant mechanisms in roots.

Cytological and proteomic analyses of horsetail (Equisetum arvense L.) spore germination.

Spermatophyte pollen tubes and root hairs have been used as single-cell-type model systems to understand the molecular processes underlying polar growth of plant cells. Horsetail (Equisetum arvense L.) is a perennial herb species in Equisetopsida, which creates separately growing spring and summer stems in its life cycle. The mature chlorophyllous spores produced from spring stems can germinate without dormancy. Here we report the cellular features and protein expression patterns in five stages of horsetail spore germination (mature spores, rehydrated spores, double-celled spores, germinated spores, and spores with protonemal cells). Using 2-DE combined with mass spectrometry, 80 proteins were found to be abundance changed upon spore germination. Among them, proteins involved in photosynthesis, protein turnover, and energy supply were over-represented. Thirteen proteins appeared as proteoforms on the gels, indicating the potential importance of post-translational modification. In addition, the dynamic changes of ascorbate peroxidase, peroxiredoxin, and dehydroascorbate reductase implied that reactive oxygen species homeostasis is critical in regulating cell division and tip-growth. The time course of germination and diverse expression patterns of proteins in photosynthesis, energy supply, lipid and amino acid metabolism indicated that heterotrophic and autotrophic metabolism were necessary in light-dependent germination of the spores. Twenty-six proteins were involved in protein synthesis, folding, and degradation, indicating that protein turnover is vital to spore germination and rhizoid tip-growth. Furthermore, the altered abundance of 14-3-3 protein, small G protein Ran, actin, and caffeoyl-CoA O-methyltransferase revealed that signaling transduction, vesicle trafficking, cytoskeleton dynamics, and cell wall modulation were critical to cell division and polar growth. These findings lay a foundation toward understanding the molecular mechanisms underlying fern spore asymmetric division and rhizoid polar growth.

Cytological and Proteomic Analyses of Osmunda cinnamomea Germinating Spores Reveal Characteristics of Fern Spore Germination and Rhizoid Tip Growth.

Fern spore is a good single-cell model for studying the sophisticated molecular networks in asymmetric cell division, differentiation, and polar growth. Osmunda cinnamomea L. var. asiatica is one of the oldest fern species with typical separate-growing trophophyll and sporophyll. The chlorophyllous spores generated from sporophyll can germinate without dormancy. In this study, the spore ultrastructure, antioxidant enzyme activities, as well as protein and gene expression patterns were analyzed in the course of spore germination at five typical stages (i.e. mature spores, rehydrated spores, double-celled spores, germinated spores, and spores with protonemal cells). Proteomic analysis revealed 113 differentially expressed proteins, which were mainly involved in photosynthesis, reserve mobilization, energy supplying, protein synthesis and turnover, reactive oxygen species scavenging, signaling, and cell structure modulation. The presence of multiple proteoforms of 25 differentially expressed proteins implies that post-translational modification may play important roles in spore germination. The dynamic patterns of proteins and their encoding genes exhibited specific characteristics in the processes of cell division and rhizoid tip growth, which include heterotrophic and autotrophic metabolisms, de novo protein synthesis and active protein turnover, reactive oxygen species and hormone (brassinosteroid and ethylene) signaling, and vesicle trafficking and cytoskeleton dynamic. In addition, the function skew of proteins in fern spores highlights the unique and common mechanisms when compared with evolutionarily divergent spermatophyte pollen. These findings provide an improved understanding of the typical single-celled asymmetric division and polar growth during fern spore germination.