ARGONAUTE proteins in cell biology and plant development. / Bialka ARGONAUT w biologii komórki i rozwoju roslin.

ARGONAUTE (AGO) proteins are integral parts of regulatory pathways under the control of small RNA (sRNA) that are fundamental for the proper functioning of eukaryotic cells. AGOs, as highly specialized platforms binding specific sRNA, coordinate gene silencing through interaction with other protein factors (forming the RNA-induced silencing complex, RISC), contributing to endonucleolytic cleavage of the target mRNA and/or influencing the translation process. The increasing number of evidence confirms the participation of AGO proteins in several other cellular processes, such as i.e.: transcription regulation, sequestration, RNA-dependent methylation of DNA, repair of DNA damages, synthesis of siRNA independent of DCL (DICER-like) proteins, or co-transcriptional regulation of MIRNA genes expression and intron splicing. Particular plant species are characterized by the presence of a different number of AGO proteins, in many cases of yet unknown regulatory and/or biological function. This review article covers the current knowledge about the functions of AGOs in cell biology and plant development.

The novel activity of Argonautes in intron splicing: A transcriptome-wide survey in plants.

The importance of the evolutionarily conserved Argonaute (AGO) proteins has been well recognized for their involvement in the RNA interference pathways. Recent discoveries in animals demonstrated that AGOs also participate in alternative splicing (AS). Motivated by the question whether the AGO proteins are also functional in RNA splicing in plants, we searched for the introns excised through an AGO-dependent manner in Arabidopsis (Arabidopsis thaliana). RNA sequencing (RNA-seq) data analysis uncovered hundreds of the introns up- or down-regulated in the ago1 and ago4 mutants, respectively. For different genes, AGOs might play either a positive or a negative role in intron excision, which was further validated by reverse transcription-polymerase chain reaction (RT-PCR). Some introns were specifically regulated by one of the AGO proteins, while some were regulated by both AGOs. Besides, a large portion of the AGO-dependent introns were organ-specifically regulated. RNA immunoprecipitation combined with high-throughput sequencing (RIP-seq) revealed that both AGOs preferentially bound to the intronic regions, supporting their high intron binding affinities. Immunoprecipitation followed by mass spectrometry (IP-MS) was performed to identify the proteins potentially interacting with the two AGOs. Six novel interactors (two interacting with AGO1 and four with both AGOs) involved in mRNA binding were uncovered, which might facilitate AGO-intron recognition. Analysis of the RNA-seq data from the rice (Oryza sativa) ago18 mutants revealed that hundreds of the introns were expressed in an AGO18-dependent manner. In summary, our results point to the novel role of the plant AGOs in intron splicing, paving a way for further studies on the mechanisms underlying AGO-mediated RNA splicing.

phyB and HY5 are Involved in the Blue Light-Mediated Alleviation of Dormancy of Arabidopsis Seeds Possibly via the Modulation of Expression of Genes Related to Light, GA, and ABA.

Light is one of the most important environmental factors regulating seed germination. It is known that light inhibits seed germination of some monocotyledonous species and that it is mostly related to the blue wavelength of the spectrum received by cryptochromes (cry). Research has also found that the red light (R) stimulates germination of dicotyledonous seeds and that this reaction involves mainly phytochromes (phy). Surprisingly, up to date, the role and the mechanism of action of blue light (BL) in seed biology of dicot plants is still very poorly understood and some questions are unexplained, e.g., whether BL plays a role in regulation of dicot seeds dormancy and/or germination? If, so what particular elements of light signaling pathway are involved in modulation of this(ese) process(es)? Also, is the BL action in regulation of dicot seeds dormancy and/or germination maybe due to changes of expression of genes related to metabolism and/or signaling of two phytohormones controlling seed-related events, such as gibberellins (GA) and abscisic acid (ABA)? To answer these intriguing questions, the combination of biological, transcriptomic, and genetic approaches was performed in this particular study. The germination tests show that freshly harvested wild type (WT) Arabidopsis thaliana Col-0 seeds are dormant and do not germinate in darkness (at 25 °C), while nondormant (after-ripened) seeds germinate well in these conditions. It is also proven that dormancy of seeds of this species is released in the presence of white and/or BL (λ = 447 nm) when placed at 25 °C. Presented here, novel results emphasize the role of BL in dormancy alleviation of dicot seeds, indicating that this wavelength of light spectrum received by phyB induces this process and that the sensitivity to this stimulus depends on the depth of seed dormancy. In addition, it is demonstrated that various elements of phy-mediated pathway can be used in response to the signal induced by BL in germinating dormant seeds of Arabidopsis. The quantitative real time PCR analysis supported by results of germination tests of WT, T-DNA insertion mutants (i.e., hy5, hfr1, and laf1) and overexpression transformants of Arabidopsis seeds (i.e., 35S:OE:HY5, 35S:OE:HYH, 35S:OE:HFR1, and 35S:OE:LAF1) revealed that the HY5 gene coding transcription factor is most probably responsible for the control of expression of genes involved in GA/ABA metabolism and/or signaling pathways during BL-dependent dormancy alleviation of Arabidopsis seeds, while biological functions of HYH and HFR1 are associated with regulation of germination. The model of BL action in regulation of dormancy alleviation and germination potential of Arabidopsis seeds is proposed.

Cellular Recycling of Proteins in Seed Dormancy Alleviation and Germination.

Each step of the seed-to-seed cycle of plant development including seed germination is characterized by a specific set of proteins. The continual renewal and/or replacement of these biomolecules are crucial for optimal plant adaptation. As proteins are the main effectors inside the cells, their levels need to be tightly regulated. This is partially achieved by specific proteolytic pathways via multicatalytic protease complexes defined as 20S and 26S proteasomes. In plants, the 20S proteasome is responsible for degradation of carbonylated proteins, while the 26S being a part of ubiquitin-proteasome pathway is known to be involved in proteolysis of phytohormone signaling regulators. On the other hand, the role of translational control of plant development is also well-documented, especially in the context of pollen tube growth and light signaling. Despite the current progress that has been made in seed biology, the sequence of cellular events that determine if the seed can germinate or not are still far from complete understanding. The role and mechanisms of regulation of proteome composition during processes occurring in the plant's photosynthetic tissues have been well-characterized since many years, but in non-photosynthetic seeds it has emerged as a tempting research task only since the last decade. This review discusses the recent discoveries providing insights into the role of protein turnover in seed dormancy alleviation, and germination, with a focus on the control of translation and proteasomal proteolysis. The presented novel data of translatome profiling in seeds highlighted that post-transcriptional regulation of germination results from a timely regulated initiation of translation. In addition, the importance of 26S proteasome in the degradation of regulatory elements of cellular signaling and that of the 20S complex in proteolysis of specific carbonylated proteins in hormonal- and light-dependent processes occurring in seeds is discussed. Based on the current knowledge the model of cellular recycling of proteins in germinating seeds is also proposed.

Phytohormones Signaling Pathways and ROS Involvement in Seed Germination.

Phytohormones and reactive oxygen species (ROS) are major determinants of the regulation of development and stress responses in plants. During life cycle of these organisms, signaling networks of plant growth regulators and ROS interact in order to render an appropriate developmental and environmental response. In plant's photosynthetic (e.g., leaves) and non-photosynthetic (e.g., seeds) tissues, enhanced and suboptimal ROS production is usually associated with stress, which in extreme cases can be lethal to cells, a whole organ or even an organism. However, controlled production of ROS is appreciated for cellular signaling. Despite the current progress that has been made in plant biology and increasing number of findings that have revealed roles of ROS and hormonal signaling in germination, some questions still arise, e.g., what are the downstream protein targets modified by ROS enabling stimulus-specific cellular responses of the seed? Or which molecular regulators allow ROS/phytohormones interactions and what is their function in seed life? In this particular review the role of some transcription factors, kinases and phosphatases is discussed, especially those which usually known to be involved in ROS and hormonal signal transduction under stress in plants, may also play a role in the regulation of processes occurring in seeds. The summarized recent findings regarding particular ROS- and phytohormones-related regulatory proteins, as well as their integration, allowed to propose a novel, possible model of action of LESION SIMULATING DISEASE 1, ENHANCED DISEASE SUSCEPTIBILITY 1, and PHYTOALEXIN DEFICIENT 4 functioning during seeds life.

Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.

Promotion of testa rupture during garden cress germination involves seed compartment-specific expression and activity of pectin methylesterases.

Pectin methylesterase (PME) controls the methylesterification status of pectins and thereby determines the biophysical properties of plant cell walls, which are important for tissue growth and weakening processes. We demonstrate here that tissue-specific and spatiotemporal alterations in cell wall pectin methylesterification occur during the germination of garden cress (Lepidium sativum). These cell wall changes are associated with characteristic expression patterns of PME genes and resultant enzyme activities in the key seed compartments CAP (micropylar endosperm) and RAD (radicle plus lower hypocotyl). Transcriptome and quantitative real-time reverse transcription-polymerase chain reaction analysis as well as PME enzyme activity measurements of separated seed compartments, including CAP and RAD, revealed distinct phases during germination. These were associated with hormonal and compartment-specific regulation of PME group 1, PME group 2, and PME inhibitor transcript expression and total PME activity. The regulatory patterns indicated a role for PME activity in testa rupture (TR). Consistent with a role for cell wall pectin methylesterification in TR, treatment of seeds with PME resulted in enhanced testa permeability and promoted TR. Mathematical modeling of transcript expression changes in germinating garden cress and Arabidopsis (Arabidopsis thaliana) seeds suggested that group 2 PMEs make a major contribution to the overall PME activity rather than acting as PME inhibitors. It is concluded that regulated changes in the degree of pectin methylesterification through CAP- and RAD-specific PME and PME inhibitor expression play a crucial role during Brassicaceae seed germination.

Embryo growth, testa permeability, and endosperm weakening are major targets for the environmentally regulated inhibition of Lepidium sativum seed germination by myrigalone A.

Myrigalone A (MyA) is a rare flavonoid in fruit leachates of Myrica gale, a deciduous shrub adapted to flood-prone habitats. As a putative allelochemical it inhibits seed germination and seedling growth. Using Lepidium sativum as a model target species, experiments were conducted to investigate how environmental cues modulate MyA's interference with key processes of seed germination. Time course analyses of L. sativum testa and endosperm rupture under different light conditions and water potentials were combined with quantifying testa permeability, endosperm weakening, tissue-specific gibberellin (GA) and abscisic acid (ABA) contents, as well as embryo growth and apoplastic superoxide production important for cell expansion growth. Lepidium sativum testa permeability and early water uptake by imbibition is enhanced by MyA. During late germination, MyA inhibits endosperm weakening and embryo growth, both processes required for endosperm rupture. Inhibition of embryo cell expansion by MyA depends on environmental cues, which is evident from the light-modulated severity of the MyA-mediated inhibition of apoplastic superoxide accumulation. Several important key weakening and growth processes during early and late germination are targets for MyA. These effects are modulated by light conditions and ambient water potential. It is speculated that MyA is a soil seed bank-destroying allelochemical that secures the persistence of M. gale in its flood-prone environment.

Myrigalone A inhibits Lepidium sativum seed germination by interference with gibberellin metabolism and apoplastic superoxide production required for embryo extension growth and endosperm rupture.

Myrica gale L. (sweet gale) fruit leachate contains myrigalone A (MyA), a rare C-methylated dihydrochalcone and putative allelochemical, which is known to be a phytotoxin impeding seedling growth. We found that MyA inhibited Lepidium sativum L. seed germination in a dose-dependent manner. MyA did not affect testa rupture, but inhibited endosperm rupture and the transition to subsequent seedling growth. MyA inhibited micropylar endosperm cap (CAP) weakening and the increase in the growth potential of the radical/hypocotyl region (RAD) of the embryo, both being key processes required for endosperm rupture. We compared the contents of abscisic acid (ABA) and gibberellins in the tissues and found that the major bioactive forms of gibberellin in L. sativum seed tissues were GA(4) and GA(6), while GA(8) and GA(13) were abundant inactive metabolites. MyA did not appreciably affect the ABA contents, but severely interfered with gibberellin metabolism and signaling by inhibiting important steps catalyzed by GA3 oxidase, as well as by interfering with the GID1-type gibberellin signaling pathway. The hormonally and developmentally regulated formation of apoplastic superoxide radicals is important for embryo growth. Specific zones within the RAD were associated with accumulation of apoplastic superoxide radicals and endoreduplication indicative of embryo cell extension. MyA negatively affected both of these processes and acted as a scavenger of apoplastic reactive oxygen species. We propose that MyA is an allelochemical with a novel mode of action on seed germination.

Regulation of seed germination in the close Arabidopsis relative Lepidium sativum: a global tissue-specific transcript analysis.

The completion of germination in Lepidium sativum and other endospermic seeds (e.g. Arabidopsis [Arabidopsis thaliana]) is regulated by two opposing forces, the growth potential of the radicle (RAD) and the resistance to this growth from the micropylar endosperm cap (CAP) surrounding it. We show by puncture force measurement that the CAP progressively weakens during germination, and we have conducted a time-course transcript analysis of RAD and CAP tissues throughout this process. We have also used specific inhibitors to investigate the importance of transcription, translation, and posttranslation levels of regulation of endosperm weakening in isolated CAPs. Although the impact of inhibiting translation is greater, both transcription and translation are required for the completion of endosperm weakening in the whole seed population. The majority of genes expressed during this process occur in both tissues, but where they are uniquely expressed, or significantly differentially expressed between tissues, this relates to the functions of the RAD as growing tissue and the CAP as a regulator of germination through weakening. More detailed analysis showed that putative orthologs of cell wall-remodeling genes are expressed in a complex manner during CAP weakening, suggesting distinct roles in the RAD and CAP. Expression patterns are also consistent with the CAP being a receptor for environmental signals influencing germination. Inhibitors of the aspartic, serine, and cysteine proteases reduced the number of isolated CAPs in which weakening developed, and inhibition of the 26S proteasome resulted in its complete cessation. This indicates that targeted protein degradation is a major control point for endosperm weakening.

The mechanisms involved in seed dormancy alleviation by hydrogen cyanide unravel the role of reactive oxygen species as key factors of cellular signaling during germination.

The physiological dormancy of sunflower (Helianthus annuus) embryos can be overcome during dry storage (after-ripening) or by applying exogenous ethylene or hydrogen cyanide (HCN) during imbibition. The aim of this work was to provide a comprehensive model, based on oxidative signaling by reactive oxygen species (ROS), for explaining the cellular mode of action of HCN in dormancy alleviation. Beneficial HCN effect on germination of dormant embryos is associated with a marked increase in hydrogen peroxide and superoxide anion generation in the embryonic axes. It is mimicked by the ROS-generating compounds methylviologen and menadione but suppressed by ROS scavengers. This increase results from an inhibition of catalase and superoxide dismutase activities and also involves activation of NADPH oxidase. However, it is not related to lipid reserve degradation or gluconeogenesis and not associated with marked changes in the cellular redox status controlled by the glutathione/glutathione disulfide couple. The expression of genes related to ROS production (NADPHox, POX, AO1, and AO2) and signaling (MAPK6, Ser/ThrPK, CaM, and PTP) is differentially affected by dormancy alleviation either during after-ripening or by HCN treatment, and the effect of cyanide on gene expression is likely to be mediated by ROS. It is also demonstrated that HCN and ROS both activate similarly ERF1, a component of the ethylene signaling pathway. We propose that ROS play a key role in the control of sunflower seed germination and are second messengers of cyanide in seed dormancy release.

Release of sunflower seed dormancy by cyanide: cross-talk with ethylene signalling pathway.

Freshly harvested sunflower (Helianthus annuus L.) seeds are considered to be dormant because they fail to germinate at relatively low temperatures (10 degrees C). This dormancy results mainly from an embryo dormancy and disappears during dry storage. Although endogenous ethylene is known to be involved in sunflower seed alleviation of dormancy, little attention had been paid to the possible role of cyanide, which is produced by the conversion of 1-aminocyclopropane 1-carboxylic acid to ethylene, in this process. The aims of this work were to investigate whether exogenous cyanide could improve the germination of dormant sunflower seeds and to elucidate its putative mechanisms of action. Naked dormant seeds became able to germinate at 10 degrees C when they were incubated in the presence of 1 mM gaseous cyanide. Other respiratory inhibitors showed that this effect did not result from an activation of the pentose phosphate pathway or the cyanide-insensitive pathway. Cyanide stimulated germination of dormant seeds in the presence of inhibitors of ethylene biosynthesis, but its improving effect required functional ethylene receptors. It did not significantly affect ethylene production and the expression of genes involved in ethylene biosynthesis or in the first steps of ethylene signalling pathway. However, the expression of the transcription factor Ethylene Response Factor 1 (ERF1) was markedly stimulated in the presence of gaseous cyanide. It is proposed that the mode of action of cyanide in sunflower seed dormancy alleviation does not involve ethylene production and that ERF1 is a common component of the ethylene and cyanide signalling pathways.

ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation.

At harvest, sunflower (Helianthus annuus L.) seeds are dormant and unable to germinate at temperatures below 15 degrees C. Seed storage in the dry state, known as after-ripening, is associated with an alleviation of embryonic dormancy allowing subsequent germination at suboptimal temperatures. To identify the process by which dormancy is broken during after-ripening, we focused on the role of reactive oxygen species (ROS) in this phenomenon. After-ripening entailed a progressive accumulation of ROS, namely superoxide anions and hydrogen peroxide, in cells of embryonic axes. This accumulation, which was investigated at the cellular level by electron microscopy, occurred concomitantly with lipid peroxidation and oxidation (carbonylation) of specific embryo proteins. Incubation of dormant seeds for 3 h in the presence of hydrogen cyanide (a compound that breaks dormancy) or methylviologen (a ROS-generating compound) also released dormancy and caused the oxidation of a specific set of embryo proteins. From these observations, we propose a novel mechanism for seed dormancy alleviation. This mechanism involves ROS production and targeted changes in protein carbonylation patterns.

Induction of oxidative stress by sunflower phytotoxins in germinating mustard seeds.

The aim of this study was to investigate the phytotoxic effect of sunflower on physiological and biochemical processes during germination of mustard seeds (Sinapis alba L. cv. Nakielska). To exclude the involvement of osmotic stress in seed reaction to phytotoxic compounds, we compared the effect of 10% (w/v) water extract from sunflower (Helianthus annuus L. cv. Ogrodowy) leaves and 28.4% (w/v) polyethylene glycol (PEG) 8000 solution characterized by an equal Psi = -1 MPa. We evaluated (1) the amount of hydrogen peroxide (H2O2); (2) activities of antioxidant enzymes: superoxide dismutase, catalase, and glutathione reductase; (3) membrane permeability; and (4) level of malondialdehyde (MDA). Both, sunflower compounds and PEG solutions inhibited mustard seed germination, but only phytotoxins caused an increase in the cell membrane permeability, MDA level, H2O2 concentration, and alterations in activities of antioxidant enzymes. Our results demonstrate that despite the activation of the antioxidant system by sunflower phytotoxins, reactive oxygen species accumulation caused cellular damage, which resulted in the decrease of germinability and gradual loss of seed vigor. It seems that the negative effect of sunflower on germination of mustard seeds is mostly because of its toxicity and not to its contribution to osmotic potential.