The DC1 Domain Protein BINUCLEATE POLLEN is Required for POLLEN Development in Arabidopsis thaliana.

The development of the male gametophyte is a tightly regulated process that requires the precise control of cell division and gene expression. A relevant aspect to understand the events underlying pollen development regulation constitutes the identification and characterization of the genes required for this process. In this work, we showed that the DC1 domain protein BINUCLEATE POLLEN (BNP) is essential for pollen development and germination. Pollen grains carrying a defective BNP alleles failed to complete mitosis II and exhibited impaired pollen germination. By yeast two-hybrid analysis and bimolecular fluorescence complementation assays, we identified a set of BNP-interacting proteins. Among confirmed interactors, we found the NAC family transcriptional regulators Vascular Plant One-Zinc Finger 1 (VOZ1) and VOZ2. VOZ1 localization changes during pollen development, moving to the vegetative nucleus at the tricellular stage. We observed that this relocalization requires BNP; in the absence of BNP in pollen from bnp/BNP plants, VOZ1 nuclear localization is impaired. As the voz1voz2 double mutants showed the same developmental defect observed in bnp pollen grains, we propose that BNP requirement to complete microgametogenesis could be linked to its interaction with VOZ1/2 proteins. BNP could have the role of a scaffold protein, recruiting VOZ1/2 to the endosomal system into assemblies that are required for their further translocation to the nucleus, where they act as transcriptional regulators.

Measuring and Perturbing Ferroptosis in Plants.

Ferroptosis is an oxidative iron-dependent cell death that was recently described in vertebrates, invertebrates, fungi, plants, and bacteria. In plants, ferroptosis has been reported in response to heat shock in roots of 6-day-old Arabidopsis thaliana seedlings. Generally, all biochemical and morphological ferroptosis hallmarks are conserved between animals and plants. Here, we describe a protocol to induce and quantify ferroptosis in plants based on the analysis of dead cells with a Sytox Green stain. Furthermore, heat shock induced cell death is prevented by using specific ferroptosis inhibitors.

A mitochondrial ADXR-ADX-P450 electron transport chain is essential for maternal gametophytic control of embryogenesis in Arabidopsis.

Mitochondrial adrenodoxins (ADXs) are small iron-sulfur proteins with electron transfer properties. In animals, ADXs transfer electrons between an adrenodoxin reductase (ADXR) and mitochondrial P450s, which is crucial for steroidogenesis. Here we show that a plant mitochondrial steroidogenic pathway, dependent on an ADXR-ADX-P450 shuttle, is essential for female gametogenesis and early embryogenesis through a maternal effect. The steroid profile of maternal and gametophytic tissues of wild-type (WT) and adxr ovules revealed that homocastasterone is the main steroid present in WT gametophytes and that its levels are reduced in the mutant ovules. The application of exogenous homocastasterone partially rescued adxr and P450 mutant phenotypes, indicating that gametophytic homocastasterone biosynthesis is affected in the mutants and that a deficiency of this hormone causes the phenotypic alterations observed. These findings also suggest not only a remarkable similarity between steroid biosynthetic pathways in plants and animals but also a common function during sexual reproduction.

Roles of cytochromes P450 in plant reproductive development.

The cytochrome P450 superfamily is a large enzymatic protein family that is widely distributed along diverse kingdoms. In plants, cytochrome P450 monooxygenases (CYPs) participate in a vast array of pathways leading to the synthesis and modification of multiple metabolites with variable and important functions during different stages of plant development. This includes the biosynthesis and degradation of a great assortment of compounds implicated in a variety of physiological responses, such as signaling and defense, organ patterning and the biosynthesis of structural polymers, among others. In this review, we summarize the characteristics of the different families of plant CYPs, focusing on the most recent advances in elucidating the roles of CYPs in plant growth and development and more specifically, during plant gametogenesis, fertilization and embryogenesis.

The MED30 subunit of mediator complex is essential for early plant development and promotes flowering in Arabidopsis thaliana.

Mediator is a large multiprotein complex that is required for the transcription of most, if not all, genes transcribed by RNA Polymerase II. A core set of subunits is essential to assemble a functional Mediator in vitro and, therefore, the corresponding loss-of-function mutants are expected to be lethal. The MED30 subunit is essential in animal systems, but is absent in yeast. Here, we report that MED30 is also essential for both male gametophyte and embryo development in the model plant Arabidopsis thaliana Mutant med30 pollen grains were viable and some were able to germinate and target the ovules, although the embryos aborted shortly after fertilization, suggesting that MED30 is important for the paternal control of early embryo development. When gametophyte defects were bypassed by specific pollen complementation, loss of MED30 led to early embryo development arrest. Later in plant development, MED30 promotes flowering through multiple signaling pathways; its downregulation led to a phase change delay, downregulation of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 (SPL3), FLOWERING LOCUS T (FTI) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), and upregulation of FLOWERING LOCUS C (FLC).

Different Types Domains are Present in Complex I from Immature Seeds and of CA Adult Plants in Arabidopsis thaliana.

Mitochondrial Nicotinamide adenine dinucleotide (NADH) dehydrogenase complex is the first complex of the mitochondrial electron transfer chain. In plants and in a variety of eukaryotes except Opisthokonta, complex I (CI) contains an extra spherical domain called carbonic anhydrase (CA) domain. This domain is thought to be composed of trimers of gamma type CA and CA-like subunits. In Arabidopsis, the CA gene family contains five members (CA1, CA2, CA3, CAL1 and CAL2). The CA domain appears to be crucial for CI assembly and is essential for normal embryogenesis. As CA and CA-like proteins are arranged in trimers to form the CA domain, it is possible for the complex to adopt different arrangements that might be tissue-specific or have specialized functions. In this work, we show that the proportion of specific CI changes in a tissue-specific manner. In immature seeds, CI assembly may be indistinctly dependent on CA1, CA2 or CA3. However, in adult plant tissues (or tissues derived from stem cells, as cell cultures), CA2-dependent CI is clearly the most abundant. This difference might account for specific physiological functions. We present evidence suggesting that CA3 does not interact with any other CA family member. As CA3 was found to interact with CI FRO1 (NDUFS4) subunit, which is located in the matrix arm, this suggests a role for CA3 in assembly and stability of CI.

Regulation of lipid peroxidation and ferroptosis in diverse species.

Lipid peroxidation is the process by which oxygen combines with lipids to generate lipid hydroperoxides via intermediate formation of peroxyl radicals. Vitamin E and coenzyme Q10 react with peroxyl radicals to yield peroxides, and then these oxidized lipid species can be detoxified by glutathione and glutathione peroxidase 4 (GPX4) and other components of the cellular antioxidant defense network. Ferroptosis is a form of regulated nonapoptotic cell death involving overwhelming iron-dependent lipid peroxidation. Here, we review the functions and regulation of lipid peroxidation, ferroptosis, and the antioxidant network in diverse species, including humans, other mammals and vertebrates, plants, invertebrates, yeast, bacteria, and archaea. We also discuss the potential evolutionary roles of lipid peroxidation and ferroptosis.

Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease.

Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis has been implicated in the pathological cell death associated with degenerative diseases (i.e., Alzheimer's, Huntington's, and Parkinson's diseases), carcinogenesis, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and kidney degeneration in mammals and is also implicated in heat stress in plants. Ferroptosis may also have a tumor-suppressor function that could be harnessed for cancer therapy. This Primer reviews the mechanisms underlying ferroptosis, highlights connections to other areas of biology and medicine, and recommends tools and guidelines for studying this emerging form of regulated cell death.

Arabidopsis phosphatidylinositol-phospholipase C2 (PLC2) is required for female gametogenesis and embryo development.

MAIN CONCLUSION: AtPLC2 is an essential gene in Arabidopsis, since it is required for female gametogenesis and embryo development. AtPLC2 might play a role in cell division during embryo-sac development and early embryogenesis. Phosphoinositide-specific phospholipase C (PI-PLC) plays an important role in signal transduction during plant development and in the response to various biotic- and abiotic stresses. The Arabidopsis PI-PLC gene family is composed of nine members, named PLC1 to PLC9. Here, we report that PLC2 is involved in female gametophyte development and early embryogenesis. Using two Arabidopsis allelic T-DNA insertion lines with different phenotypic penetrations, we observed both female gametophytic defects and aberrant embryos. For the plc2-1 mutant (Ws background), no homozygous plants could be recovered in the offspring from self-pollinated plants. Nonetheless, plc2-1 hemizygous mutants are affected in female gametogenesis, showing embryo sacs arrested at early developmental stages. Allelic hemizygous plc2-2 mutant plants (Col-0 background) present reduced seed set and embryos arrested at the pre-globular stage with abnormal patterns of cell division. A low proportion (0.8%) of plc2-2 homozygous mutants was found to escape lethality and showed morphological defects and disrupted megagametogenesis. PLC2-promoter activity was observed during early megagametogenesis, and after fertilization in the embryo proper. Immunolocalization studies in early stage embryos revealed that PLC2 is restricted to the plasma membrane. Altogether, these results establish a role for PLC2 in both reproductive- and embryo development, presumably by controlling mitosis and/or the formation of cell-division planes.

Functional characterization of mutants affected in the carbonic anhydrase domain of the respiratory complex I in Arabidopsis thaliana.

The NADH-ubiquinone oxidoreductase complex (complex I) (EC 1.6.5.3) is the main entrance site of electrons into the respiratory chain. In a variety of eukaryotic organisms, except animals and fungi (Opisthokonta), it contains an extra domain comprising trimers of putative γ-carbonic anhydrases, named the CA domain, which has been proposed to be essential for assembly of complex I. However, its physiological role in plants is not fully understood. Here, we report that Arabidopsis mutants defective in two CA subunits show an altered photorespiratory phenotype. Mutants grown in ambient air show growth retardation compared to wild-type plants, a feature that is reversed by cultivating plants in a high-CO2 atmosphere. Moreover, under photorespiratory conditions, carbon assimilation is diminished and glycine accumulates, suggesting an imbalance with respect to photorespiration. Additionally, transcript levels of specific CA subunits are reduced in plants grown under non-photorespiratory conditions. Taken together, these results suggest that the CA domain of plant complex I contributes to sustaining efficient photosynthesis under ambient (photorespiratory) conditions.

Auxin Import and Local Auxin Biosynthesis Are Required for Mitotic Divisions, Cell Expansion and Cell Specification during Female Gametophyte Development in Arabidopsis thaliana.

The female gametophyte of flowering plants, called the embryo sac, develops from a haploid cell named the functional megaspore, which is specified after meiosis by the diploid sporophyte. In Arabidopsis, the functional megaspore undergoes three syncitial mitotic divisions followed by cellularization to form seven cells of four cell types including two female gametes. The plant hormone auxin is important for sporophytic developmental processes, and auxin levels are known to be regulated by biosynthesis and transport. Here, we investigated the role of auxin biosynthetic genes and auxin influx carriers in embryo sac development. We find that genes from the YUCCA/TAA pathway (YUC1, YUC2, YUC8, TAA1, TAR2) are expressed asymmetrically in the developing ovule and embryo sac from the two-nuclear syncitial stage until cellularization. Mutants for YUC1 and YUC2 exhibited defects in cell specification, whereas mutations in YUC8, as well as mutations in TAA1 and TAR2, caused defects in nuclear proliferation, vacuole formation and anisotropic growth of the embryo sac. Additionally, expression of the auxin influx carriers AUX1 and LAX1 were observed at the micropylar pole of the embryo sac and in the adjacent cells of the ovule, and the aux1 lax1 lax2 triple mutant shows multiple gametophyte defects. These results indicate that both localized auxin biosynthesis and auxin import, are required for mitotic divisions, cell expansion and patterning during embryo sac development.

Auxin-dependent patterning and gamete specification in the Arabidopsis female gametophyte.

The female reproductive unit of flowering plants, the haploid female gametophyte, is highly reduced relative to other land plants. We show that patterning of the Arabidopsis female gametophyte depends on an asymmetric distribution of the hormone auxin during its syncitial development. Furthermore, this auxin gradient is correlated with location-specific auxin biosynthesis, rather than auxin efflux that directs patterning in the diploid sporophytic tissues comprising the rest of the plant. Manipulation of auxin responses or synthesis induces switching of gametic and nongametic cell identities and specialized nonreproductive cells to exhibit attributes presumptively lost during angiosperm evolution. These findings may account for the unique egg cell specification characteristic of angiosperms and the formation of seeds with single diploid embryos while containing endosperm that can have variable numbers of parental haploid genomes.

A collection of Ds insertional mutants associated with defects in male gametophyte development and function in Arabidopsis thaliana.

Functional analyses of the Arabidopsis genome require analysis of the gametophytic generation, since approximately 10% of the genes are expressed in the male gametophyte and approximately 9% in the female gametophyte. Here we describe the genetic and molecular characterization of 67 Ds insertion lines that show reduced transmission through the male gametophyte. About half of these mutations are male gametophytic-specific mutations, while the others also affect female transmission. Genomic sequences flanking both sides of the Ds element were recovered for 39 lines; for 16 the Ds elements were inserted in or close to coding regions, while 7 were located in intergenic/unannotated regions of the genome. For the remaining 16 lines, chromosomal rearrangements such as translocations or deletions, ranging between 30 and 500 kb, were associated with the transposition event. The mutants were classified into five groups according to the developmental processes affected; these ranged from defects in early stages of gametogenesis to later defects affecting pollen germination, pollen tube growth, polarity or guidance, or pollen tube-embryo sac interactions or fertilization. The isolated mutants carry Ds insertions in genes with diverse biological functions and potentially specify new functions for several unannotated or unknown proteins.

Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis.

The plant life cycle involves an alternation of generations between sporophyte and gametophyte. Currently, the genes and pathways involved in gametophytic development and function in flowering plants remain largely unknown. A large-scale mutant screen of Ds transposon insertion lines was employed to identify 130 mutants of Arabidopsis thaliana with defects in female gametophyte development and function. A wide variety of mutant phenotypes were observed, ranging from defects in different stages of early embryo sac development to mutants with apparently normal embryo sacs, but exhibiting defects in processes such as pollen tube guidance, fertilization or early embryo development. Unexpectedly, nearly half of the mutants isolated in this study were found to be primarily defective in post-fertilization processes dependent on the maternal allele, suggesting that genes expressed from the female gametophyte or the maternal genome play a major role in the early development of plant embryos. Sequence identification of the genes disrupted in the mutants revealed genes involved in protein degradation, cell death, signal transduction and transcriptional regulation required for embryo sac development, fertilization and early embryogenesis. These results provide a first comprehensive overview of the genes and gene products involved in female gametophyte development and function within a flowering plant.

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.