Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin.

Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light accumulated diatoxanthin to diadinoxanthin in low-light in diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin-diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.

Reduced physiological plasticity in a fish adapted to stable temperatures.

Plasticity can allow organisms to maintain consistent performance across a wide range of environmental conditions. However, it remains largely unknown how costly plasticity is and whether a trade-off exists between plasticity and performance under optimal conditions. Biological rates generally increase with temperature, and to counter that effect, fish use physiological plasticity to adjust their biochemical and physiological functions. Zebrafish in the wild encounter large daily and seasonal temperature fluctuations, suggesting they should display high physiological plasticity. Conversely, laboratory zebrafish have been at optimal temperatures with low thermal fluctuations for over 150 generations. We treated this domestication as an evolution experiment and asked whether this has reduced the physiological plasticity of laboratory fish compared to their wild counterparts. We measured a diverse range of phenotypic traits, from gene expression through physiology to behavior, in wild and laboratory zebrafish acclimated to 15 temperatures from 10 °C to 38 °C. We show that adaptation to the laboratory environment has had major effects on all levels of biology. Laboratory fish show reduced plasticity and are thus less able to counter the direct effects of temperature on key traits like metabolic rates and thermal tolerance, and this difference is detectable down to gene expression level. Rapid selection for faster growth in stable laboratory environments appears to have carried with it a trade-off against physiological plasticity in captive zebrafish compared with their wild counterparts.

Author Correction: The Seminavis robusta genome provides insights into the evolutionary adaptations of benthic diatoms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Seminavis robusta genome provides insights into the evolutionary adaptations of benthic diatoms.

Benthic diatoms are the main primary producers in shallow freshwater and coastal environments, fulfilling important ecological functions such as nutrient cycling and sediment stabilization. However, little is known about their evolutionary adaptations to these highly structured but heterogeneous environments. Here, we report a reference genome for the marine biofilm-forming diatom Seminavis robusta, showing that gene family expansions are responsible for a quarter of all 36,254 protein-coding genes. Tandem duplications play a key role in extending the repertoire of specific gene functions, including light and oxygen sensing, which are probably central for its adaptation to benthic habitats. Genes differentially expressed during interactions with bacteria are strongly conserved in other benthic diatoms while many species-specific genes are strongly upregulated during sexual reproduction. Combined with re-sequencing data from 48 strains, our results offer insights into the genetic diversity and gene functions in benthic diatoms.

The Myb-like transcription factor phosphorus starvation response (PtPSR) controls conditional P acquisition and remodelling in marine microalgae.

Phosphorus (P) is one of the limiting macronutrients for algal growth in marine environments. Microalgae have developed adaptation mechanisms to P limitation that involve remodelling of internal phosphate resources and accumulation of lipids. Here, we used in silico analyses to identify the P-stress regulator PtPSR (Phaeodactylum tricornutum phosphorus starvation response) in the diatom P. tricornutum. ptpsr mutant lines were generated using gene editing and characterised by various molecular, genetics and biochemical tools. PtPSR belongs to a clade of Myb transcription factors that are conserved in stramenopiles and distantly related to plant P-stress regulators. PtPSR bound specifically to a conserved cis-regulatory element found in the regulatory region of P-stress-induced genes. ptpsr knockout mutants showed reduction in cell growth under P limitation. P-stress responses were impaired in ptpsr mutants compared with wild-type, including reduced induction of P-stress response genes, near to complete loss of alkaline phosphatase activity and reduced phospholipid degradation. We conclude that PtPSR is a key transcription factor influencing P scavenging, phospholipid remodelling and cell growth in adaptation to P stress in diatoms.

PAMP-INDUCED SECRETED PEPTIDE 3 modulates immunity in Arabidopsis.

Small post-translationally modified peptides are important signalling components of plant defence responses against phytopathogens, acting as both positive and negative modulators. PAMP-INDUCED SECRETED PEPTIDE (PIP) 1 and 2 have been shown to amplify plant immunity. Here we investigate the role of the related peptide PIP3 in the regulation of immune response in Arabidopsis. Treatment with synthetic PIP peptides led to similar transcriptome reprogramming, indicating an effect on innate immunity-related processes and phytohormones, including jasmonic acid (JA) biosynthesis and signalling. PIP3 overexpressing (OX) plants showed enhanced growth inhibition in response to flg22 exposure. In addition, flg22-induced production of reactive oxygen species and callose deposition was significantly reduced in PIP3-OX plants. Interestingly, PIP3-OX plants showed increased susceptibility toward both Botrytis cinerea and the biotrophic pathogen Pseudomonas syringae. Expression of both JA and salicylic acid (SA) biosynthesis and signalling genes was more induced during B. cinerea infection in PIP3-OX plants compared with wild-type plants. Promoter and ChIP-seq analyses indicated that the transcription factors WRKY18, WRKY33, and WRKY40 cooperatively act as repressors for PIP3. The results point to a fine-tuning role for PIP3 in modulation of immunity through the regulation of SA and JA biosynthesis and signalling pathways in Arabidopsis.

Molecular adaptations to phosphorus deprivation and comparison with nitrogen deprivation responses in the diatom Phaeodactylum tricornutum.

Phosphorus, an essential element for all living organisms, is a limiting nutrient in many regions of the ocean due to its fast recycling. Changes in phosphate (Pi) availability in aquatic systems affect diatom growth and productivity. We investigated the early adaptive mechanisms in the marine diatom Phaeodactylum tricornutum to P deprivation using a combination of transcriptomics, metabolomics, physiological and biochemical experiments. Our analysis revealed strong induction of gene expression for proteins involved in phosphate acquisition and scavenging, and down-regulation of processes such as photosynthesis, nitrogen assimilation and nucleic acid and ribosome biosynthesis. P deprivation resulted in alterations of carbon allocation through the induction of the pentose phosphate pathway and cytosolic gluconeogenesis, along with repression of the Calvin cycle. Reorganization of cellular lipids was indicated by coordinated induced expression of phospholipases, sulfolipid biosynthesis enzymes and a putative betaine lipid biosynthesis enzyme. A comparative analysis of nitrogen- and phosphorus-deprived P. tricornutum revealed both common and distinct regulation patterns in response to phosphate and nitrate stress. Regulation of central carbon metabolism and amino acid metabolism was similar, whereas unique responses were found in nitrogen assimilation and phosphorus scavenging in nitrogen-deprived and phosphorus-deprived cells, respectively.

The effects of phosphorus limitation on carbon metabolism in diatoms.

Phosphorus is an essential element for life, serving as an integral component of nucleic acids, lipids and a diverse range of other metabolites. Concentrations of bioavailable phosphorus are low in many aquatic environments. Microalgae, including diatoms, apply physiological and molecular strategies such as phosphorus scavenging or recycling as well as adjusting cell growth in order to adapt to limiting phosphorus concentrations. Such strategies also involve adjustments of the carbon metabolism. Here, we review the effect of phosphorus limitation on carbon metabolism in diatoms. Two transcriptome studies are analysed in detail, supplemented by other transcriptome, proteome and metabolite data, to gain an overview of different pathways and their responses. Phosphorus, nitrogen and silicon limitation responses are compared, and similarities and differences discussed. We use the current knowledge to propose a suggestive model for the carbon flow in phosphorus-replete and phosphorus-limited diatom cells.This article is part of the themed issue 'The peculiar carbon metabolism in diatoms'.

Dynamic responses to silicon in Thalasiossira pseudonana - Identification, characterisation and classification of signature genes and their corresponding protein motifs.

The diatom cell wall, or frustule, is a highly complex, three-dimensional structure consisting of nanopatterned silica as well as proteins and other organic components. While some key components have been identified, knowledge on frustule biosynthesis is still fragmented. The model diatom Thalassiosira pseudonana was subjected to silicon (Si) shift-up and shift-down situations. Cellular and molecular signatures, dynamic changes and co-regulated clusters representing the hallmarks of cellular and molecular responses to changing Si availabilities were characterised. Ten new proteins with silaffin-like motifs, two kinases and a novel family of putatively frustule-associated transmembrane proteins induced by Si shift-up with a possible role in frustule biosynthesis were identified. A separate cluster analysis performed on all significantly regulated silaffin-like proteins (SFLPs), as well as silaffin-like motifs, resulted in the classification of silaffins, cingulins and SFLPs into distinct clusters. A majority of the genes in the Si-responsive clusters are highly divergent, but positive selection does not seem to be the driver behind this variability. This study provides a high-resolution map over transcriptional responses to changes in Si availability in T. pseudonana. Hallmark Si-responsive genes are identified, characteristic motifs and domains are classified, and taxonomic and evolutionary implications outlined and discussed.

The IDA-LIKE peptides IDL6 and IDL7 are negative modulators of stress responses in Arabidopsis thaliana.

Small signalling peptides have emerged as important cell to cell messengers in plant development and stress responses. However, only a few of the predicted peptides have been functionally characterized. Here, we present functional characterization of two members of the IDA-LIKE (IDL) peptide family in Arabidopsis thaliana, IDL6 and IDL7. Localization studies suggest that the peptides require a signal peptide and C-terminal processing to be correctly transported out of the cell. Both IDL6 and IDL7 appear to be unstable transcripts under post-transcriptional regulation. Treatment of plants with synthetic IDL6 and IDL7 peptides resulted in down-regulation of a broad range of stress-responsive genes, including early stress-responsive transcripts, dominated by a large group of ZINC FINGER PROTEIN (ZFP) genes, WRKY genes, and genes encoding calcium-dependent proteins. IDL7 expression was rapidly induced by hydrogen peroxide, and idl7 and idl6 idl7 double mutants displayed reduced cell death upon exposure to extracellular reactive oxygen species (ROS). Co-treatment of the bacterial elicitor flg22 with IDL7 peptide attenuated the rapid ROS burst induced by treatment with flg22 alone. Taken together, our results suggest that IDL7, and possibly IDL6, act as negative modulators of stress-induced ROS signalling in Arabidopsis.

A sex-inducing pheromone triggers cell cycle arrest and mate attraction in the diatom Seminavis robusta.

Although sexual reproduction is believed to play a major role in the high diversification rates and species richness of diatoms, a mechanistic understanding of diatom life cycle control is virtually lacking. Diatom sexual signalling is controlled by a complex, yet largely unknown, pheromone system. Here, a sex-inducing pheromone (SIP(+)) of the benthic pennate diatom Seminavis robusta was identified by comparative metabolomics, subsequently purified, and physicochemically characterized. Transcriptome analysis revealed that SIP(+) triggers the switch from mitosis-to-meiosis in the opposing mating type, coupled with the transcriptional induction of proline biosynthesis genes, and the release of the proline-derived attraction pheromone. The induction of cell cycle arrest by a pheromone, chemically distinct from the one used to attract the opposite mating type, highlights the existence of a sophisticated mechanism to increase chances of mate finding, while keeping the metabolic losses associated with the release of an attraction pheromone to a minimum.

Whole-cell response to nitrogen deprivation in the diatom Phaeodactylum tricornutum.

Algal growth is strongly affected by nitrogen (N) availability. Diatoms, an ecologically important group of unicellular algae, have evolved several acclimation mechanisms to cope with N deprivation. In this study, we integrated physiological data with transcriptional and metabolite data to reveal molecular and metabolic modifications in N-deprived conditions in the marine diatom Phaeodactylum tricornutum. Physiological and metabolite measurements indicated that the photosynthetic capacity and chlorophyll content of the cells decreased, while neutral lipids increased in N-deprived cultures. Global gene expression analysis showed that P. tricornutum responded to N deprivation through an increase in N transport, assimilation, and utilization of organic N resources. Following N deprivation, reduced biosynthesis and increased recycling of N compounds like amino acids, proteins, and nucleic acids was observed at the transcript level. The majority of the genes associated with photosynthesis and chlorophyll biosynthesis were also repressed. Carbon metabolism was restructured through downregulation of the Calvin cycle and chrysolaminarin biosynthesis, and co-ordinated upregulation of glycolysis, the tricarboxylic acid cycle, and pyruvate metabolism, leading to funnelling of carbon sources to lipid metabolism. Finally, reallocation of membrane lipids and induction of de novo triacylglycerol biosynthesis directed cells to accumulation of neutral lipids.

The IDA/IDA-LIKE and PIP/PIP-LIKE gene families in Arabidopsis: phylogenetic relationship, expression patterns, and transcriptional effect of the PIPL3 peptide.

Peptide ligands play crucial roles in the life cycle of plants by modulating the innate immunity against pathogens and regulating growth and developmental processes. One well-studied example is INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), which controls floral organ abscission and lateral root emergence in Arabidopsis thaliana. IDA belongs to a family of five additional IDA-LIKE (IDL) members that have all been suggested to be involved in regulation of Arabidopsis development. Here we present three novel members of the IDL subfamily and show that two of them are strongly and rapidly induced by different biotic and abiotic stresses. Furthermore, we provide data that the recently identified PAMP-INDUCED SECRETED PEPTIDE (PIP) and PIP-LIKE (PIPL) peptides, which show similarity to the IDL and C-TERMINALLY ENCODED PEPTIDE (CEP) peptides, are not only involved in innate immune response in Arabidopsis but are also induced by abiotic stress. Expression patterns of the IDA/IDL and PIP/PIPL genes were analysed using in silico data, qRT-PCR and GUS promoter lines. Transcriptomic responses to PIPL3 peptide treatment suggested a role in regulation of biotic stress responses and cell wall modification.

System responses to equal doses of photosynthetically usable radiation of blue, green, and red light in the marine diatom Phaeodactylum tricornutum.

Due to the selective attenuation of solar light and the absorption properties of seawater and seawater constituents, free-floating photosynthetic organisms have to cope with rapid and unpredictable changes in both intensity and spectral quality. We have studied the transcriptional, metabolic and photo-physiological responses to light of different spectral quality in the marine diatom Phaeodactylum tricornutum through time-series studies of cultures exposed to equal doses of photosynthetically usable radiation of blue, green and red light. The experiments showed that short-term differences in gene expression and profiles are mainly light quality-dependent. Transcription of photosynthesis-associated nuclear genes was activated mainly through a light quality-independent mechanism likely to rely on chloroplast-to-nucleus signaling. In contrast, genes encoding proteins important for photoprotection and PSII repair were highly dependent on a blue light receptor-mediated signal. Changes in energy transfer efficiency by light-harvesting pigments were spectrally dependent; furthermore, a declining trend in photosynthetic efficiency was observed in red light. The combined results suggest that diatoms possess a light quality-dependent ability to activate photoprotection and efficient repair of photodamaged PSII. In spite of approximately equal numbers of PSII-absorbed quanta in blue, green and red light, the spectral quality of light is important for diatom responses to ambient light conditions.

The chloroplast genome of the diatom Seminavis robusta: new features introduced through multiple mechanisms of horizontal gene transfer.

The chloroplasts of heterokont algae such as diatoms are the result of a secondary endosymbiosis event, in which a red alga was engulfed by a non-photosynthetic eukaryote. The diatom chloroplast genomes sequenced to date show a high degree of similarity, but some examples of gene replacement or introduction of genes through horizontal gene transfer are known. The evolutionary origin of the gene transfers is unclear. We have sequenced and characterised the complete chloroplast genome and a putatively chloroplast-associated plasmid of the pennate diatom Seminavis robusta. The chloroplast genome contains two introns, a feature that has not previously been found in diatoms. The group II intron of atpB appears to be recently transferred from a Volvox-like green alga. The S. robusta chloroplast genome (150,905 bp) is the largest diatom chloroplast genome characterised to date, mainly due to the presence of four large gene-poor regions. Open reading frames (ORFs) encoded by the gene-poor regions show similarity to putative proteins encoded by the chloroplast genomes of different heterokonts, as well as the plasmids pCf1 and pCf2 found in the diatom Cylindrotheca fusiformis. A tyrosine recombinase and a serine recombinase are encoded by the S. robusta chloroplast genome, indicating a possible mechanism for the introduction of novel genes. A plasmid with similarity to pCf2 was also identified. Phylogenetic analyses of three ORFs identified on pCf2 suggest that two of them are part of an operon-like gene cluster conserved in bacteria. Several genetic elements have moved through horizontal gene transfer between the chloroplast genomes of different heterokonts. Two recombinases are likely to promote such gene insertion events, and the plasmid identified may act as vectors in this process. The copy number of the plasmid was similar to that of the plastid genome indicating a plastid localization.

NEVERSHED and INFLORESCENCE DEFICIENT IN ABSCISSION are differentially required for cell expansion and cell separation during floral organ abscission in Arabidopsis thaliana.

Floral organ shedding is a cell separation event preceded by cell-wall loosening and generally accompanied by cell expansion. Mutations in NEVERSHED (NEV) or INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) block floral organ abscission in Arabidopsis thaliana. NEV encodes an ADP-ribosylation factor GTPase-activating protein, and cells of nev mutant flowers display membrane-trafficking defects. IDA encodes a secreted peptide that signals through the receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2). Analyses of single and double mutants revealed unique features of the nev and ida phenotypes. Cell-wall loosening was delayed in ida flowers. In contrast, nev and nev ida mutants displayed ectopic enlargement of abscission zone (AZ) cells, indicating that cell expansion alone is not sufficient to trigger organ loss. These results suggest that NEV initially prevents precocious cell expansion but is later integral for cell separation. IDA is involved primarily in the final cell separation step. A mutation in KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1), a suppressor of the ida mutant, could not rescue the abscission defects of nev mutant flowers, indicating that NEV-dependent activity downstream of KNAT1 is required. Transcriptional profiling of mutant AZs identified gene clusters regulated by IDA-HAE/HSL2. Several genes were more strongly downregulated in nev-7 compared with ida and hae hsl2 mutants, consistent with the rapid inhibition of organ loosening in nev mutants, and the overlapping roles of NEV and IDA in cell separation. A model of the crosstalk between the IDA signalling pathway and NEV-mediated membrane traffic during floral organ abscission is presented.

Molecular and photosynthetic responses to prolonged darkness and subsequent acclimation to re-illumination in the diatom Phaeodactylum tricornutum.

Photosynthetic diatoms that live suspended throughout the water column will constantly be swept up and down by vertical mixing. When returned to the photic zone after experiencing longer periods in darkness, mechanisms exist that enable the diatoms both to survive sudden light exposure and immediately utilize the available energy in photosynthesis and growth. We have investigated both the response to prolonged darkness and the re-acclimation to moderate intensity white irradiance (E = 100 µmol m(-2) s(-1)) in the diatom Phaeodactylum tricornutum, using an integrated approach involving global transcriptional profiling, pigment analyses, imaging and photo-physiological measurements. The responses were studied during continuous white light, after 48 h of dark treatment and after 0.5 h, 6 h, and 24 h of re-exposure to the initial irradiance. The analyses resulted in several intriguing findings. Dark treatment of the cells led to 1) significantly decreased nuclear transcriptional activity, 2) distinct intracellular changes, 3) fixed ratios of the light-harvesting pigments despite a decrease in the total cell pigment pool, and 4) only a minor drop in photosynthetic efficiency (Φ(PSII_max)). Re-introduction of the cells to the initial light conditions revealed 5) distinct expression profiles for nuclear genes involved in photosynthesis and those involved in photoprotection, 6) rapid rise in photosynthetic parameters (α and rETR(max)) within 0.5 h of re-exposure to light despite a very modest de novo synthesis of photosynthetic compounds, and 7) increasingly efficient resonance energy transfer from fucoxanthin chlorophyll a/c-binding protein complexes to photosystem II reaction centers during the first 0.5 h, supporting the observations stated in 6). In summary, the results show that despite extensive transcriptional, metabolic and intracellular changes, the ability of cells to perform photosynthesis was kept intact during the length of the experiment. We conclude that P. tricornutum maintains a functional photosynthetic apparatus during dark periods that enables prompt recovery upon re-illumination.

Gene regulation of carbon fixation, storage, and utilization in the diatom Phaeodactylum tricornutum acclimated to light/dark cycles.

The regulation of carbon metabolism in the diatom Phaeodactylum tricornutum at the cell, metabolite, and gene expression levels in exponential fed-batch cultures is reported. Transcriptional profiles and cell chemistry sampled simultaneously at all time points provide a comprehensive data set on carbon incorporation, fate, and regulation. An increase in Nile Red fluorescence (a proxy for cellular neutral lipids) was observed throughout the light period, and water-soluble glucans increased rapidly in the light period. A near-linear decline in both glucans and lipids was observed during the dark period, and transcription profile data indicated that this decline was associated with the onset of mitosis. More than 4,500 transcripts that were differentially regulated during the light/dark cycle are identified, many of which were associated with carbohydrate and lipid metabolism. Genes not previously described in algae and their regulation in response to light were integrated in this analysis together with proposed roles in metabolic processes. Some very fast light-responding genes in, for example, fatty acid biosynthesis were identified and allocated to biosynthetic processes. Transcripts and cell chemistry data reflect the link between light energy availability and light energy-consuming metabolic processes. Our data confirm the spatial localization of processes in carbon metabolism to either plastids or mitochondria or to glycolysis/gluconeogenesis, which are localized to the cytosol, chloroplast, and mitochondria. Localization and diel expression pattern may be of help to determine the roles of different isoenzymes and the mining of genes involved in light responses and circadian rhythms.

Genome-wide profiling of responses to cadmium in the diatom Phaeodactylum tricornutum.

The only group of organisms in which a biological function for cadmium has been shown is the diatoms, which are unicellular phytoplankton. Yet diatoms exhibit similar sensitivity to Cd as other groups of phytoplankton. We have investigated responses of Cd on molecular, metabolic, and physiological levels in the diatom Phaeodactylum tricornutum. P. tricornutum apparently has a high tolerance to Cd; only minor responses were observed on growth, pigment, and transcriptional changes at cadmium concentrations of 123 µg/L. No significant changes in chlorophyll and xanthophyll levels were observed, and the very few transcripts affected strongly indicate that the cells were able to respond to the increased Cd(2+) levels without changing proteins levels. At 10 times this concentration, 1230 µg/L, a much clearer response was observed, including transcripts encoding proteins involved in metal transport, cell signaling, and detoxification processes. Our results point toward putative pathways for the removal or detoxification of Cd and its metabolites as well as a possible Cd uptake mechanism. We predict that ATPase5-1B is involved in removal of Cd by pumping Cd(2+) ions out of the cell, whereas VIT1/CCC1 sequesters Cd(2+) in the vacuole.

Arabidopsis thaliana MIRO1 and MIRO2 GTPases are unequally redundant in pollen tube growth and fusion of polar nuclei during female gametogenesis.

MIRO GTPases have evolved to regulate mitochondrial trafficking and morphology in eukaryotic organisms. A previous study showed that T-DNA insertion in the Arabidopsis MIRO1 gene is lethal during embryogenesis and affects pollen tube growth and mitochondrial morphology in pollen, whereas T-DNA insertion in MIRO2 does not affect plant development visibly. Phylogenetic analysis of MIRO from plants revealed that MIRO 1 and 2 orthologs in dicots cluster in two separate groups due to a gene/genome duplication event, suggesting that functional redundancy may exists between the two MIRO genes. To investigate this possibility, we generated miro1(+/-)/miro2-2(-/-) plants. Compared to miro1(+/-) plants, the miro1(+/-)/miro2-2(-/-) plants showed increased segregation distortion. miro1(+/-)/miro2-2(-/-) siliques contained less aborted seeds, but more than 3 times the number of undeveloped ovules. In addition, reciprocal crosses showed that co-transmission through the male gametes was nearly absent, whereas co-transmission through the female gametes was severely reduced in miro1(+/-)/miro2-2(-/-) plants. Further investigations revealed that loss of MIRO2 (miro2(-/-)) function in the miro1(+/-) background enhanced pollen tube growth defects. In developing miro1(+/-)/miro2(-/-) embryo sacs, fusion of polar nuclei was further delayed or impaired compared to miro1 plants. This phenotype has not been reported previously for miro1 plants and coincides with studies showing that defects in some mitochondria-targeted genes results in the same phenotype. Our observations show that loss of function in MIRO2 in a miro1(+/-) background enhances the miro1(+/-) phenotype significantly, even though miro2(-/-) plants alone does not display any phenotypes. Based on these findings, we conclude that MIRO1 and MIRO2 are unequally redundant and that a proportion of the miro1(+/-)/miro2(-/-) plants haploid gametes displays the complete null phenotype of MIRO GTPase function at key developmental stages.