oiwa,a Female Gametophytic Mutant Impaired in a Mitochondrial Manganese-Superoxide Dismutase,Reveals Crucial Roles for Reactive Oxygen Species during Embryo Sac Development and Fertilization in Arabidopsis

Reactive oxygen species (ROS) can function as signaling molecules, regulating key aspects of plant development, or as toxic compounds leading to oxidative damage. In this article, we show that the regulation of ROS production during megagametogenesis is largely dependent on MSD1, a mitochondrial Mn-superoxide dismutase. Wild-type mature embryo sacs show ROS exclusively in the central cell, which appears to be the main source of ROS before pollination. Accordingly, MSD1 shows a complementary expression pattern. MSD1 expression is elevated in the egg apparatus at maturity but is downregulated in the central cell. The oiwa mutants are characterized by high levels of ROS detectable in both the central cell and the micropylar cells. Remarkably, egg apparatus cells in oiwa show central cell features, indicating that high levels of ROS result in the expression of central cell characteristic genes. Notably, ROS are detected in synergid cells after pollination. This ROS burst depends on stigma pollination but precedes fertilization, suggesting that embryo sacs sense the imminent arrival of pollen tubes and respond by generating an oxidative environment. Altogether, we show that ROS play a crucial role during female gametogenesis and fertilization. MSD1 activity seems critical for maintaining ROS localization and important for embryo sac patterning.     

New insights into the functional roles of reactive oxygen species during embryo sac development and fertilization in Arabidopsis thaliana

Previously considered as toxic by-products of aerobic metabolism, reactive oxygen species (ROS) are emerging as essential signaling molecules in eukaryotes. Recent evidence showed that maintenance of ROS homeostasis during female gametophyte development is crucial for embryo sac patterning and fertilization. Although ROS are exclusively detected in the central cell of mature embryo sacs, the study of mutants deficient in ROS homeostasis suggests that controlled oxidative bursts might take place earlier during gametophyte development. Also, a ROS burst that depends on pollination takes place inside the embryo sac. This oxidative response might be required for pollen tube growth arrest and for sperm cell release. In this mini-review, we will focus on new insights into the role of ROS during female gametophyte development and fertilization. Special focus will be made on the mitochondrial Mn-Superoxide dismutase (MSD1), which has been recently reported to be essential for maintaining ROS homeostasis during embryo sac formation.     

Extracellular sunflower proteins: evidence on non-classical secretion of a jacalin-related lectin

Extracellular proteins from sunflower seedlings were analyzed by electrophoresis followed by peptide mass fingerprinting. Tentative identification revealed novel proteins for this crop. A significant number of those proteins were not expected to be extracellular because they lacked the typical signal peptide responsible for secretion. In silico analysis showed that some members of this group presented the characteristic disordered structures of certain non-classical and leaderless mammalian secretory proteins. Among these proteins, a putative jacalin-related lectin (Helja) with a mannose binding domain was further isolated from extracellular fluids by mannose-affinity chromatography, thus validating its identification. Besides, immunolocalization assays confirmed its extracellular location. These results showed that a lectin, not predicted to be secreted in strict requirement of the N-terminal signal peptide, occurs in a sunflower extracellular compartment. The implications of this finding are discussed.     

On the role of a Lipid-Transfer Protein. Arabidopsis ltp3 mutant is compromised in germination and seedling growth.

Plant Lipid-Transfer Proteins (LTPs) exhibit the ability to reversibly bind/transport lipids in vitro. LTPs have been involved in diverse physiological processes but conclusive evidence on their role has only been presented for a few members, none of them related to seed physiology. Arabidopsis seeds rely on storage oil breakdown to supply carbon skeletons and energy for seedling growth. Here, Arabidopsis ltp3 mutant was analyzed for its ability to germinate and for seedling establishment. Ltp3 showed delayed germination and reduced germination frequency. Seedling growth appeared reduced in the mutant but this growth restriction was rescued by the addition of an exogenous carbon supply, suggesting a defective oil mobilization. Lipid breakdown analysis during seedling growth revealed a differential profile in the mutant compared to the wild type. The involvement of LTP3 in germination and seedling growth and its relationship with the lipid transfer ability of this protein is discussed.     

Nitric oxide and cyclic GMP are messengers in the indole acetic acid-induced adventitious rooting process

This report describes part of the signaling pathway and some of the molecules involved in the auxin-induced adventitious root formation in cucumber (Cucumis sativus). Previous results showed that nitric oxide (NO) mediates the auxin response during adventitious root formation (Pagnussat et al., 2002). To determine the order of action of indole acetic acid (IAA) and NO within the signal transduction pathway and to elucidate the target molecules that are downstream of NO action, cucumber hypocotyl cuttings were submitted to a pretreatment leading to endogenous auxin depletion. The auxin depletion treatment provoked a 3-fold reduction of the root number in comparison to the nondepleted explants. The NO-donor sodium nitroprusside was able to promote adventitious rooting in auxin-depleted explants, whereas the specific NO scavenger cPTIO prevented the effect of sodium nitroprusside. The endogenous NO level was monitored in both control and auxin-depleted explants using a NO-specific fluorescent probe. The NO level was 3.5-fold higher in control (nondepleted) explants than in auxin-depleted ones. The exogenous application of IAA restored the NO concentration to the level found in nondepleted explants. Because NO activates the enzyme guanylate cyclase (GC), we analyzed the involvement of the messenger cGMP in the adventitious root development mediated by IAA and NO. The GC inhibitor LY83583 reduced root development induced by IAA and NO, whereas the cell-permeable cGMP derivative 8-Br-cGMP reversed this effect. The endogenous level of cGMP is regulated by both the synthesis via GC and its degradation by the phosphodiesterase activity. When assayed, the phosphodiesterase inhibitor sildenafil citrate was able to induce adventitious rooting in both nondepleted and auxin-depleted explants. Results indicate that NO operates downstream of IAA promoting adventitious root development through the GC-catalyzed synthesis of cGMP.     

A CDPK type protein kinase is involved in rice SPS light modulation

A protein kinase activity that can phosphorylate and inactivate rice ( Oryza sativa ) sucrose-phosphate synthase (SPS; UDP-glucose: d -fructose-6-phosphate-2-glucosyl transferase, EC 2.4.1.14) was measured in extracts prepared from leaves exposed to light-dark transitions. Enzyme activity present in extracts from dark leaves was about 5-fold higher than the activity in extracts from leaves that had been collected in the light. The protein kinase (named R-SPSK) was purified about 100-fold from dark leaves and its biochemical properties were studied. The micromolar dependence of Ca²⁺ exhibited by R-SPSK, and its response to calmodulin antagonists was similar to the properties associated with members of the plant Calcium-Dependent Protein Kinase (CDPK) family. Two modulators of SPS activity, Pi and Glc-6-P, were examined for an effect on R-SPSK. While Glc-6-P did not affect R-SPSK activity, Pi drastically increased the kinase activity. Taken together, these data provide evidence that SPS may be regulated by a CDPK type protein-kinase whose activity is modulated by light-dark transitions and stimulated by Pi, the negative effector of SPS activity.     

C-ferroptosis is an iron-dependent form of regulated cell death in cyanobacteria

Ferroptosis is an oxidative and iron-dependent form of regulated cell death (RCD) recently described in eukaryotic organisms like animals, plants, and parasites. Here, we report that a similar process takes place in the photosynthetic prokaryote Synechocystis sp. PCC 6803 in response to heat stress. After a heat shock, Synechocystis sp. PCC 6803 cells undergo a cell death pathway that can be suppressed by the canonical ferroptosis inhibitors, CPX, vitamin E, Fer-1, liproxstatin-1, glutathione (GSH), or ascorbic acid (AsA). Moreover, as described for eukaryotic ferroptosis, this pathway is characterized by an early depletion of the antioxidants GSH and AsA, and by lipid peroxidation. These results indicate that all of the hallmarks described for eukaryotic ferroptosis are conserved in photosynthetic prokaryotes and suggest that ferroptosis might be an ancient cell death program.     

Cell-fate switch of synergid to egg cell in Arabidopsis eostre mutant embryo sacs arises from misexpression of the BEL1-like homeodomain gene BLH1

In Arabidopsis thaliana, the female gametophyte is a highly polarized structure consisting of four cell types: one egg cell and two synergids, one central cell, and three antipodal cells. In this report, we describe the characterization of a novel female gametophyte mutant, eostre, which affects establishment of cell fates in the mature embryo sac. The eostre phenotype is caused by misexpression of the homeodomain gene BEL1-like homeodomain 1 (BLH1) in the embryo sac. It is known that BELL-KNAT proteins function as heterodimers whose activities are regulated by the Arabidopsis ovate family proteins (OFPs). We show that the phenotypic effect of BLH1 overexpression is dependent upon the class II knox gene KNAT3, suggesting that KNAT3 must be expressed and functional during megagametogenesis. Moreover, disruption of At OFP5, a known interactor of KNAT3 and BLH1, partially phenocopies the eostre mutation. Our study indicates that suppression of ectopic activity of BELL-KNOX TALE complexes, which might be mediated by At OFP5, is essential for normal development and cell specification in the Arabidopsis embryo sac. As eostre-1 embryo sacs also show nuclear migration abnormalities, this study suggests that a positional mechanism might be directing establishment of cell fates in early megagametophyte development.     

Rapid endocytosis is triggered upon imbibition in Arabidopsis seeds

During seed imbibition and embryo activation, rapid change from a metabolically resting state to the activation of diverse extracellular and/or membrane bound molecules is essential and, hence, endocytosis could be activated too. In fact, we have documented endocytic internalization of the membrane impermeable endocytic tracer FM4–64 already upon 30 min of imbibition of Arabidopsis seeds. This finding suggest that endocytosis is activated early during seed imbibition in Arabidopsis. Immunolocalization of rhamnogalacturonan-II (RG-II) complexed with boron showed that whereas this pectin is localized only in the cell walls of dry seed embryos, it starts to be intracellular once the imbibition started. Brefeldin A (BFA) exposure resulted in recruitment of the intracellular RG-II pectin complexes into the endocytic BFA-induced compartments, confirming the endocytic origin of the RG-II signal detected intracellularly. Finally, germination was significantly delayed when Arabidopsis seeds were germinated in the presence of inhibitors of endocytic pathways, suggesting that trafficking of extracellular molecules might play an important role in the overcome of germination. This work constitutes the first demonstration of endocytic processes during germination and opens new perspectives about the role of the extracellular matrix and membrane components in seed germination.