Perspectives on embryo maturation and seed quality in a global climate change scenario.

Global climate change has already brought noticeable alterations to multiple regions of our planet. Several important steps of plant growth and development, such as embryogenesis, can be affected by environmental changes. For instance, these changes would affect how stored nutrients are used during early stages of seed germination as it transitions from a heterotrophic to autotrophic metabolism, a critical period for the seedling's survival. In this perspective, we provide a brief description of relevant processes that occur during embryo maturation and account for nutrient accumulation, which are sensitive to environmental change. As examples of the effects associated with climate change are increased CO2 levels and changes in temperature. During seed development, most of the nutrients stored in the seed are accumulated during the seed maturation stage. These nutrients include, depending on the plant species, carbohydrates, lipids and proteins. Regarding micronutrients, it has also been established that iron, a key micronutrient for various electron transfer processes in plant cells, accumulates during embryo maturation. Several articles have been published indicating that climate change can affect the quality of the seed, in terms of total nutritional content, but also, it may affect seed production. Here we discuss the potential effects of temperature and CO2 increase from an embryo autonomous point of view, in an attempt to separate the maternal effects from embryonic effects.

Using an embryo specific promoter to modify iron distribution pattern in Arabidopsis.

Iron is an essential micronutrient for life. During the development of the seed, iron accumulates during embryo maturation. In Arabidopsis thaliana, iron mainly accumulates in the vacuoles of only one cell type, the cell layer that surrounds provasculature in hypocotyl and cotyledons. Iron accumulation pattern in Arabidopsis is an exception in plant phylogeny, most part of the dicot embryos accumulate iron in several cell layers including cortex and, in some cases, even in protodermis. It remains unknown how does iron reach the internal cell layers of the embryo, and in particular, the molecular mechanisms responsible of this process. Here, we use transgenic approaches to modify the iron accumulation pattern in an Arabidopsis model. Using the SDH2-3 embryo-specific promoter, we were able to express VIT1 ectopically in both a wild type background and a mutant vit1 background lacking expression of this vacuolar iron transporter. These manipulations modify the iron distribution pattern in Arabidopsis from one cell layer to several cell layers, including protodermis, cortex cells, and the endodermis. Interestingly, total seed iron content was not modified compared with the wild type, suggesting that iron distribution in embryos is not involved in the control of the total iron amount accumulated in seeds. This experimental model can be used to study the processes involved in iron distribution patterning during embryo maturation and its evolution in dicot plants.

Perls/DAB Staining to Examine Iron Distribution in Arabidopsis Embryos.

Iron is accumulated in Arabidopsis embryos during seed maturation. Where iron localizes in seed and embryo is important information for seed research. Iron detection can be performed in an inexpensive manner using Perls staining, based on the Prussian blue complex formation. After this first step, DAB intensification can be performed in order to visualize easily where iron pools are located in isolated embryos.

Growth Developmental Defects of Mitochondrial Iron Transporter 1 and 2 Mutants in Arabidopsis in Iron Sufficient Conditions.

Iron is the most abundant micronutrient in plant mitochondria, and it has a crucial role in biochemical reactions involving electron transfer. It has been described in Oryza sativa that Mitochondrial Iron Transporter (MIT) is an essential gene and that knockdown mutant rice plants have a decreased amount of iron in their mitochondria, strongly suggesting that OsMIT is involved in mitochondrial iron uptake. In Arabidopsis thaliana, two genes encode MIT homologues. In this study, we analyzed different AtMIT1 and AtMIT2 mutant alleles, and no phenotypic defects were observed in individual mutant plants grown in normal conditions, confirming that neither AtMIT1 nor AtMIT2 are individually essential. When we generated crosses between the Atmit1 and Atmit2 alleles, we were able to isolate homozygous double mutant plants. Interestingly, homozygous double mutant plants were obtained only when mutant alleles of Atmit2 with the T-DNA insertion in the intron region were used for crossings, and in these cases, a correctly spliced AtMIT2 mRNA was generated, although at a low level. Atmit1 Atmit2 double homozygous mutant plants, knockout for AtMIT1 and knockdown for AtMIT2, were grown and characterized in iron-sufficient conditions. Pleiotropic developmental defects were observed, including abnormal seeds, an increased number of cotyledons, a slow growth rate, pinoid stems, defects in flower structures, and reduced seed set. A RNA-Seq study was performed, and we could identify more than 760 genes differentially expressed in Atmit1 Atmit2. Our results show that Atmit1 Atmit2 double homozygous mutant plants misregulate genes involved in iron transport, coumarin metabolism, hormone metabolism, root development, and stress-related response. The phenotypes observed, such as pinoid stems and fused cotyledons, in Atmit1 Atmit2 double homozygous mutant plants may suggest defects in auxin homeostasis. Unexpectedly, we observed a possible phenomenon of T-DNA suppression in the next generation of Atmit1 Atmit2 double homozygous mutant plants, correlating with increased splicing of the AtMIT2 intron containing the T-DNA and the suppression of the phenotypes observed in the first generation of the double mutant plants. In these plants with a suppressed phenotype, no differences were observed in the oxygen consumption rate of isolated mitochondria; however, the molecular analysis of gene expression markers, AOX1a, UPOX, and MSM1, for mitochondrial and oxidative stress showed that these plants express a degree of mitochondrial perturbation. Finally, we could establish by a targeted proteomic analysis that a protein level of 30% of MIT2, in the absence of MIT1, is enough for normal plant growth under iron-sufficient conditions.

B3 Transcription Factors Determine Iron Distribution and FERRITIN Gene Expression in Embryo but Do Not Control Total Seed Iron Content.

Iron is an essential micronutrient for humans and other organisms. Its deficiency is one of the leading causes of anemia worldwide. The world health organization has proposed that an alternative to increasing iron content in food is through crop biofortification. One of the most consumed part of crops is the seed, however, little is known about how iron accumulation in seed occurs and how it is regulated. B3 transcription factors play a critical role in the accumulation of storage compounds such as proteins and lipids. Their role in seed maturation has been well characterized. However, their relevance in accumulation and distribution of micronutrients like iron remains unknown. In Arabidopsis thaliana and other plant models, three master regulators belonging to the B3 transcription factors family have been identified: FUSCA3 (FUS3), LEAFY COTYLEDON2 (LEC2), and ABSCISIC ACID INSENSITIVE 3 (ABI3). In this work, we studied how seed iron homeostasis is affected in B3 transcription factors mutants using histological and molecular approaches. We determined that iron distribution is modified in abi3, lec2, and fus3 embryo mutants. For abi3-6 and fus3-3 mutant embryos, iron was less accumulated in vacuoles of cells surrounding provasculature compared with wild type embryos. lec2-1 embryos showed no difference in the pattern of iron distribution in hypocotyl, but a dramatic decrease of iron was observed in cotyledons. Interestingly, for the three mutant genotypes, total iron content in dry mutant seeds showed no difference compared to wild type. At the molecular level, we showed that genes encoding the iron storage ferritins proteins are misregulated in mutant seeds. Altogether our results support a role of the B3 transcription factors ABI3, LEC2, and FUS3 in maintaining iron homeostasis in Arabidopsis embryos.

2000 years of agriculture in the Atacama desert lead to changes in the distribution and concentration of iron in maize.

We performed a histological and quantitative study of iron in archaeological maize seeds from prehispanic times recovered from Tarapacá, Atacama Desert. Also, we examined iron distribution changes at the cell level in embryos from ancient versus new varieties of maize. Our results show a progressive decrease in iron concentration from the oldest maize to modern specimens. We interpret the results as an effect of prehispanic agriculture over the micronutrient composition of maize.

Thermal effects vary predictably across levels of organization: empirical results and theoretical basis.

Thermal performance curves have provided a common framework to study the impact of temperature in biological systems. However, few generalities have emerged to date. Here, we combine an experimental approach with theoretical analyses to demonstrate that performance curves are expected to vary predictably with the levels of biological organization. We measured rates of enzymatic reactions, organismal performance and population viability in Drosophila acclimated to different thermal conditions and show that performance curves become narrower with thermal optima shifting towards lower temperatures at higher levels or organization. We then explain these results on theoretical grounds, showing that this pattern reflects the cumulative impact of asymmetric thermal effects that piles up with complexity. These results and the proposed framework are important to understand how organisms, populations and ecological communities might respond to changing thermal conditions.

Transcriptional Regulation of Iron Distribution in Seeds: A Perspective.

Several transcription factors have been involved in the regulation of gene expression during seed development. Nutritional reserves, including iron, are principally accumulated during seed maturation stages. Using the model plant Arabidopsis thaliana, it has been shown that iron is stored during seed development in vacuoles of the endodermis cell layer. During seed germination, these iron reserves are remobilized and used by the seedling during the heterotrophic to autotrophic metabolism switch. To date, no information about how iron distribution is genetically regulated has been reported.

The PAP/SAL1 retrograde signaling pathway is involved in iron homeostasis.

KEY MESSAGE: There is a link between PAP/SAL retrograde pathway, ethylene signaling and Fe metabolism in Arabidopsis. Nuclear gene expression is regulated by a diversity of retrograde signals that travel from organelles to the nucleus in a lineal or classical model. One such signal molecule is 3'-phosphoadenisine-5'-phosphate (PAP) and it's in vivo levels are regulated by SAL1/FRY1, a phosphatase enzyme located in chloroplast and mitochondria. This metabolite inhibits the action of a group of exorribonucleases which participate in post-transcriptional gene expression regulation. Transcriptome analysis of Arabidopsis thaliana mutant plants in PAP-SAL1 pathway revealed that the ferritin genes AtFER1, AtFER3, and AtFER4 are up-regulated. In this work we studied Fe metabolism in three different mutants of the PAP/SAL1 retrograde pathway. Mutant plants showed increased Fe accumulation in roots, shoots and seeds when grown in Fe-sufficient condition, and a constitutive activation of the Strategy I Fe uptake genes. As a consequence, they grew more vigorously than wild type plants in Fe-deficient medium. However, when mutant plants grown in Fe-deficient conditions were sprayed with Fe in their leaves, they were unable to deactivate root Fe uptake. Ethylene synthesis inhibition revert the constitutive Fe uptake phenotype. We propose that there is a link between PAP/SAL pathway, ethylene signaling and Fe metabolism.

The Transcription Factor bHLH121 Interacts with bHLH105 (ILR3) and Its Closest Homologs to Regulate Iron Homeostasis in Arabidopsis.

Iron (Fe) is an essential micronutrient for plant growth and development. Any defects in the maintenance of Fe homeostasis will alter plant productivity and the quality of their derived products. In Arabidopsis (Arabidopsis thaliana), the transcription factor ILR3 plays a central role in controlling Fe homeostasis. In this study, we identified bHLH121 as an ILR3-interacting transcription factor. Interaction studies showed that bHLH121 also interacts with the three closest homologs of ILR3 (i.e., basic-helix-loop-helix 34 [bHLH34], bHLH104, and bHLH115). bhlh121 loss-of-function mutants displayed severe defects in Fe homeostasis that could be reverted by exogenous Fe supply. bHLH121 acts as a direct transcriptional activator of key genes involved in the Fe regulatory network, including bHLH38, bHLH39, bHLH100, bHLH101, POPEYE, BRUTUS, and BRUTUS LIKE1, as well as IRONMAN1 and IRONMAN2 In addition, bHLH121 is necessary for activating the expression of transcription factor gene FIT in response to Fe deficiency via an indirect mechanism. bHLH121 is expressed throughout the plant body, and its expression is not affected by Fe availability. By contrast, Fe availability affects the cellular localization of bHLH121 protein in roots. Altogether, these data show that bHLH121 is a regulator of Fe homeostasis that acts upstream of FIT in concert with ILR3 and its closest homologs.

Transcriptional integration of the responses to iron availability in Arabidopsis by the bHLH factor ILR3.

Iron (Fe) homeostasis is crucial for all living organisms. In mammals, an integrated posttranscriptional mechanism couples the regulation of both Fe deficiency and Fe excess responses. Whether in plants an integrated control mechanism involving common players regulates responses both to deficiency and to excess is still to be determined. In this study, molecular, genetic and biochemical approaches were used to investigate transcriptional responses to both Fe deficiency and excess. A transcriptional activator of responses to Fe shortage in Arabidopsis, called bHLH105/ILR3, was found to also negatively regulate the expression of ferritin genes, which are markers of the plant's response to Fe excess. Further investigations revealed that ILR3 repressed the expression of several structural genes that function in the control of Fe homeostasis. ILR3 interacts directly with the promoter of its target genes, and repressive activity was conferred by its dimerisation with bHLH47/PYE. Last, this study highlighted that important facets of plant growth in response to Fe deficiency or excess rely on ILR3 activity. Altogether, the data presented herein support that ILR3 is at the centre of the transcriptional regulatory network that controls Fe homeostasis in Arabidopsis, in which it acts as both transcriptional activator and repressor.

The Diverse Iron Distribution in Eudicotyledoneae Seeds: From Arabidopsis to Quinoa.

Seeds accumulate iron during embryo maturation stages of embryogenesis. Using Arabidopsis thaliana as model plant, it has been described that mature embryos accumulate iron within a specific cell layer, the endodermis. This distribution pattern was conserved in most of the analyzed members from Brassicales, with the exception of the basal Vasconcellea pubescens that also showed elevated amounts of iron in cortex cells. To determine whether the V. pubescens iron distribution was indicative of a wider pattern in non-Brassicales Eudicotyledoneae, we studied iron distribution pattern in different embryos belonging to plant species from different Orders from Eudicotyledoneae and one basal from Magnoliidae. The results obtained indicate that iron distribution in A. thaliana embryo is an extreme case of apomorphic character found in Brassicales, not-extensive to the rest of Eudicotyledoneae.

Increasing Provasculature Complexity in the Arabidopsis Embryo May Increase Total Iron Content in Seeds: A Hypothesis.

Anemia due to iron deficiency is a worldwide issue, affecting mainly children and women. Seed iron is a major source of this micronutrient for feeding, however, in most crops these levels are too low to meet daily needs. Thus, increasing iron allocation and its storage in seeds can represent an important step to enhance iron provision for humans and animals. Our knowledge on seed iron homeostasis is mainly based on studies performed in the model plant Arabidopsis thaliana, where iron accumulates in endodermis cells surrounding the embryo provasculature. It has been reported that cotyledon provasculature pattern complexity can be modified, thus we hypothesize that changes in the complexity of embryo vein patterns may affect total iron content in Arabidopsis seeds. This approach could be used as basis to develop strategies aimed to biofortify seeds.

Dynamic Subcellular Localization of Iron during Embryo Development in Brassicaceae Seeds.

Iron is an essential micronutrient for plants. Little is know about how iron is loaded in embryo during seed development. In this article we used Perls/DAB staining in order to reveal iron localization at the cellular and subcellular levels in different Brassicaceae seed species. In dry seeds of Brassica napus, Nasturtium officinale, Lepidium sativum, Camelina sativa, and Brassica oleracea iron localizes in vacuoles of cells surrounding provasculature in cotyledons and hypocotyl. Using B. napus and N. officinale as model plants we determined where iron localizes during seed development. Our results indicate that iron is not detectable by Perls/DAB staining in heart stage embryo cells. Interestingly, at torpedo development stage iron localizes in nuclei of different cells type, including integument, free cell endosperm and almost all embryo cells. Later, iron is detected in cytoplasmic structures in different embryo cell types. Our results indicate that iron accumulates in nuclei in specific stages of embryo maturation before to be localized in vacuoles of cells surrounding provasculature in mature seeds.

The endosomal protein CHARGED MULTIVESICULAR BODY PROTEIN1 regulates the autophagic turnover of plastids in Arabidopsis.

Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents.

The VASCULATURE COMPLEXITY AND CONNECTIVITY gene encodes a plant-specific protein required for embryo provasculature development.

The molecular mechanisms by which vascular tissues acquire their identities are largely unknown. Here, we report on the identification and characterization of VASCULATURE COMPLEXITY AND CONNECTIVITY (VCC), a member of a 15-member, plant-specific gene family in Arabidopsis (Arabidopsis thaliana) that encodes proteins of unknown function with four predicted transmembrane domains. Homozygous vcc mutants displayed cotyledon vein networks of reduced complexity and disconnected veins. Similar disconnections or gaps were observed in the provasculature of vcc embryos, indicating that defects in vein connectivity appear early in mutant embryo development. Consistently, the overexpression of VCC leads to an unusually high proportion of cotyledons with high-complexity vein networks. Neither auxin distribution nor the polar localization of the auxin efflux carrier were affected in vcc mutant embryos. Expression of VCC was detected in developing embryos and procambial, cambial, and vascular cells of cotyledons, leaves, roots, hypocotyls, and anthers. To evaluate possible genetic interactions with other genes that control vasculature patterning in embryos, we generated a double mutant for VCC and OCTOPUS (OPS). The vcc ops double mutant embryos showed a complete loss of high-complexity vascular networks in cotyledons and a drastic increase in both provascular and vascular disconnections. In addition, VCC and OPS interact physically, suggesting that VCC and OPS are part of a complex that controls cotyledon vascular complexity.

A novel endosomal sorting complex required for transport (ESCRT) component in Arabidopsis thaliana controls cell expansion and development.

ESCRT proteins mediate membrane remodeling and scission events and are essential for endosomal sorting of plasma membrane proteins for degradation. We have identified a novel, plant-specific ESCRT component called PROS (POSITIVE REGULATOR OF SKD1) in Arabidopsis thaliana. PROS has a strong positive effect on the in vitro ATPase activity of SKD1 (also known as Vacuolar Protein Sorting 4 or VPS4), a critical component required for ESCRT-III disassembly and endosomal vesiculation. PROS interacts with both SKD1 and the SKD1-positive regulator LIP5/VTA1. We have identified a putative MIM domain within PROS that mediate the interaction with the MIT domain of SKD1. Interestingly, whereas MIM domains are commonly found at the C terminus of ESCRT-III subunits, the PROS MIM domain is internal. The heterologous expression of PROS in yeast mutant cells lacking Vta1p partially rescues endosomal sorting defects. PROS is expressed in most tissues and cells types in Arabidopsis thaliana. Silencing of PROS leads to reduced cell expansion and abnormal organ growth.