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The autoinhibited plasma membrane calcium ATPase ACA8 from A. thaliana has an N-terminal autoinhibitory domain. Binding of calcium-loaded calmodulin at two sites located at residues 42-62 and 74-96 relieves autoinhibition of ACA8 activity. Through activity studies and a yeast complementation assay we investigated wild-type (WT) and N-terminally truncated ACA8 constructs (Δ20, Δ30, Δ35, Δ37, Δ40, Δ74 and Δ100) to explore the role of conserved motifs in the N-terminal segment preceding the calmodulin binding sites. Furthermore, we purified WT, Δ20- and Δ100-ACA8, tested activity in vitro and performed structural studies of purified Δ20-ACA8 stabilized in a lipid nanodisc to explore the mechanism of autoinhibition. We show that an N-terminal segment between residues 20 and 35 including conserved Phe32, upstream of the calmodulin binding sites, is important for autoinhibition and the activation by calmodulin. Cryo-EM structure determination at 3.3 Å resolution of a beryllium fluoride inhibited E2 form, and at low resolution for an E1 state combined with AlphaFold prediction provide a model for autoinhibition, consistent with the mutational studies.
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Most high-yielding crops are susceptible to abiotic and biotic stresses, making them particularly vulnerable to the potential effects of climate change. A possible alternative is to accelerate the domestication of wild plants that are already tolerant to harsh conditions and to increase their yields by methods such as gene editing. We foresee that crops' wild progenitors could potentially compete with the resulting de novo domesticated plants, reducing yields. To improve the recognition of weeds, we propose using gene editing techniques to introduce traits into de novo domesticated crops that will allow for visual recognition of the crops by weeding robots that have been trained by machine learning.
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Climate change may result in a drier climate and increased salinization, threatening agricultural productivity worldwide. Quinoa (Chenopodium quinoa) produces highly nutritious seeds and tolerates abiotic stresses such as drought and high salinity, making it a promising future food source. However, the presence of antinutritional saponins in their seeds is an undesirable trait. We mapped genes controlling seed saponin content to a genomic region that includes TSARL1. We isolated desired genetic variation in this gene by producing a large mutant library of a commercial quinoa cultivar and screening the library for specific nucleotide substitutions using droplet digital PCR. We were able to rapidly isolate two independent tsarl1 mutants, which retained saponins in the leaves and roots for defence, but saponins were undetectable in the seed coat. We further could show that TSARL1 specifically controls seed saponin biosynthesis in the committed step after 2,3-oxidosqualene. Our work provides new important knowledge on the function of TSARL1 and represents a breakthrough for quinoa breeding.
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Chenopodium quinoa , Genotipo , Saponinas , Semillas , Chenopodium quinoa/genética , Chenopodium quinoa/metabolismo , Saponinas/biosíntesis , Saponinas/metabolismo , Semillas/genética , Semillas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
P4 ATPases (i.e., lipid flippases) are eukaryotic enzymes that transport lipids across membrane bilayers. In plants, P4 ATPases are named Aminophospholipid ATPases (ALAs) and are organized into five phylogenetic clusters. Here we generated an Arabidopsis mutant lacking all five cluster-2 ALAs (ala8/9/10/11/12), which is the most highly expressed ALA subgroup in vegetative tissues. Plants harboring the quintuple knockout (KO) show rosettes that are 2.2-fold smaller and display chlorotic lesions. A similar but less severe phenotype was observed in an ala10/11 double KO. The growth and lesion phenotypes of ala8/9/10/11/12 mutants were reversed by expressing a NahG transgene, which encodes an enzyme that degrades salicylic acid (SA). A role for SA in promoting the lesion phenotype was further supported by quantitative PCR assays showing increased mRNA abundance for an SA-biosynthesis gene ISOCHORISMATE SYNTHASE 1 (ICS1) and two SA-responsive genes PATHOGENESIS-RELATED GENE 1 (PR1) and PR2. Lesion phenotypes were also reversed by growing plants in liquid media containing either low calcium (~0.1 mM) or high nitrogen concentrations (~24 mM), which are conditions known to suppress SA-dependent autoimmunity. Yeast-based fluorescent lipid uptake assays revealed that ALA10 and ALA11 display overlapping substrate specificities, including the transport of LysoPC signaling lipids. Together, these results establish that the biochemical functions of ALA8-12 are at least partially overlapping, and that deficiencies in cluster-2 ALAs result in an SA-dependent autoimmunity phenotype that has not been observed for flippase mutants with deficiencies in other ALA clusters.
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Proteínas de Arabidopsis , Arabidopsis , Ácido Salicílico/metabolismo , Filogenia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Adenosina Trifosfatasas/genética , LípidosRESUMEN
Databases of genome sequences are growing exponentially, but, in some cases, assembly is incomplete and genes are poorly annotated. For evolutionary studies, it is important to identify all members of a given gene family in a genome. We developed a method for identifying most, if not all, members of a gene family from raw genomes in which assembly is of low quality, using the P-type ATPase superfamily as an example. The method is based on the translation of an entire genome in all six reading frames and the co-occurrence of two family-specific sequence motifs that are in close proximity to each other. To test the method's usability, we first used it to identify P-type ATPase members in the high-quality annotated genome of barley (Hordeum vulgare). Subsequently, after successfully identifying plasma membrane H+-ATPase family members (P3A ATPases) in various plant genomes of varying quality, we tested the hypothesis that the number of P3A ATPases correlates with the ability of the plant to tolerate saline conditions. In 19 genomes of glycophytes and halophytes, the total number of P3A ATPase genes was found to vary from 7 to 22, but no significant difference was found between the two groups. The method successfully identified P-type ATPase family members in raw genomes that are poorly assembled.
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Hordeum , ATPasas Tipo P , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Genoma de Planta , ATPasas Tipo P/genética , Hordeum/genética , Hordeum/metabolismo , FilogeniaRESUMEN
The aerial surfaces of quinoa (Chenopodium quinoa) and common ice plant (Mesembryanthemum crystallinum) are covered with a layer of epidermal bladder cells (EBCs), which are modified non-glandular trichomes previously considered to be key to the extreme salt and drought tolerance of these plants. Here, however, we find that EBCs of these plants play only minor roles, if any, in abiotic stress tolerance and in fact are detrimental under conditions of water deficit. We report that EBCs instead function as deterrents to a broad range of generalist arthropod herbivores, through their combined function of forming both a chemical and a physical barrier, and they also serve a protective function against a phytopathogen. Our study overturns current models that link EBCs to salt and drought tolerance and assigns new functions to these structures that might provide novel possibilities for protecting crops from arthropod pests.
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Herbivoria , Vejiga Urinaria , Cloruro de Sodio , Plantas , Mecanismos de DefensaRESUMEN
P-type ATPases constitute a large ancient super-family of primary active pumps that have diverse substrate specificities ranging from H+ to phospholipids. The significance of these enzymes in biology cannot be overstated. They are structurally related, and their catalytic cycles alternate between high- and low-affinity conformations that are induced by phosphorylation and dephosphorylation of a conserved aspartate residue. In the year 1988, all P-type sequences available by then were analyzed and five major families, P1 to P5, were identified. Since then, a large body of knowledge has accumulated concerning the structure, function, and physiological roles of members of these families, but only one additional family, P6 ATPases, has been identified. However, much is still left to be learned. For each family a few remaining enigmas are presented, with the intention that they will stimulate interest in continued research in the field. The review is by no way comprehensive and merely presents personal views with a focus on evolution.
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ATPasas Tipo P , Adenosina Trifosfatasas/metabolismo , ATPasas Tipo P/metabolismoRESUMEN
Perennial grains have potential to contribute to ecological intensification of food production by enabling the direct harvest of human-edible crops without requiring annual cycles of disturbance and replanting. Studies of prototype perennial grains and other herbaceous perennials point to the ability of agroecosystems including these crops to protect water quality, enhance wildlife habitat, build soil quality, and sequester soil carbon. However, genetic improvement of perennial grain candidates has been hindered by limited investment due to uncertainty about whether the approach is viable. As efforts to develop perennial grain crops have expanded in past decades, critiques of the approach have arisen. With a recent report of perennial rice producing yields equivalent to those of annual rice over eight consecutive harvests, many theoretical concerns have been alleviated. Some valid questions remain over the timeline for new crop development, but we argue these may be mitigated by implementation of recent technological advances in crop breeding and genetics such as low-cost genotyping, genomic selection, and genome editing. With aggressive research investment in the development of new perennial grain crops, they can be developed and deployed to provide atmospheric greenhouse gas reductions.
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Agricultura , Fitomejoramiento , Humanos , Grano Comestible , Productos Agrícolas , SueloRESUMEN
Eukaryotic plasma membranes (PMs) are energized by electrogenic P-type ATPases that generate either Na+ or H+ motive forces to drive Na+ and H+ dependent transport processes, respectively. For this purpose, animal rely on Na+/K+-ATPases whereas fungi and plants employ PM H+-ATPases. Prokaryotes, on the other hand, depend on H+ or Na+-motive electron transport complexes to energize their cell membranes. This raises the question as to why and when electrogenic Na+ and H+ pumps evolved? Here it is shown that prokaryotic Na+/K+-ATPases have near perfect conservation of binding sites involved in coordination of three Na+ and two K+ ions. Such pumps are rare in Eubacteria but are common in methanogenic Archaea where they often are found together with P-type putative PM H+-ATPases. With some exceptions, Na+/K+-ATPases and PM H+-ATPases are found everywhere in the eukaryotic tree of life, but never together in animals, fungi and land plants. It is hypothesized that Na+/K+-ATPases and PM H+-ATPases evolved in methanogenic Archaea to support the bioenergetics of these ancestral organisms, which can utilize both H+ and Na+ as energy currencies. Both pumps must have been simultaneously present in the first eukaryotic cell, but during diversification of the major eukaryotic kingdoms, and at the time animals diverged from fungi, animals kept Na+/K+-ATPases but lost PM H+-ATPases. At the same evolutionary branch point, fungi did loose Na+/K+-ATPases, and their role was taken over by PM H+-ATPases. An independent but similar scenery emerged during terrestrialization of plants: they lost Na+/K+-ATPases but kept PM H+-ATPases.
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ATPasas de Translocación de Protón , ATPasa Intercambiadora de Sodio-Potasio , Animales , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , ATPasas de Translocación de Protón/metabolismo , Bombas de Protones/metabolismo , Membrana Celular/metabolismo , Eucariontes , Archaea/genética , Hongos/metabolismo , Plantas/metabolismoRESUMEN
With the appearance of plants and animals, new challenges emerged. These multicellular eukaryotes had to solve for example the difficulties of multifaceted communication between cells and adaptation to new habitats. In this paper, we are looking for one piece of the puzzle that made the development of complex multicellular eukaryotes possible with a focus on regulation of P2B autoinhibited Ca2+-ATPases. P2B ATPases pump Ca2+ out of the cytosol at the expense of ATP hydrolysis, and thereby maintain a steep gradient between the extra- and intracytosolic compartments which is utilized for Ca2+-mediated rapid cell signaling. The activity of these enzymes is regulated by a calmodulin (CaM)-responsive autoinhibitory region, which can be located in either termini of the protein, at the C-terminus in animals and at the N-terminus in plants. When the cytoplasmic Ca2+ level reaches a threshold, the CaM/Ca2+ complex binds to a calmodulin-binding domain (CaMBD) in the autoinhibitor, which leads to the upregulation of pump activity. In animals, protein activity is also controlled by acidic phospholipids that bind to a cytosolic portion of the pump. Here, we analyze the appearance of CaMBDs and the phospholipid-activating sequence and show that their evolution in animals and plants was independent. Furthermore, we hypothesize that different causes may have initiated the appearance of these regulatory layers: in animals, it is linked to the appearance of multicellularity, while in plants it co-occurs with their water-to-land transition.
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Adenosina Trifosfatasas , Calmodulina , Animales , Calmodulina/metabolismo , Unión Proteica , Señalización del Calcio , Calcio/metabolismoRESUMEN
Plasma membrane (PM) H+-ATPases are the electrogenic proton pumps that export H+ from plant and fungal cells to acidify the surroundings and generate a membrane potential. Plant PM H+-ATPases are equipped with a Cterminal autoinhibitory regulatory (R) domain of about 100 amino acid residues, which could not be identified in the PM H+-ATPases of green algae but appeared fully developed in immediate streptophyte algal predecessors of land plants. To explore the physiological significance of this domain, we created in vivo C-terminal truncations of autoinhibited PM H+ATPase2 (AHA2), one of the two major isoforms in the land plant Arabidopsis thaliana. As more residues were deleted, the mutant plants became progressively more efficient in proton extrusion, concomitant with increased expansion growth and nutrient uptake. However, as the hyperactivated AHA2 also contributed to stomatal pore opening, which provides an exit pathway for water and an entrance pathway for pests, the mutant plants were more susceptible to biotic and abiotic stresses, pathogen invasion and water loss, respectively. Taken together, our results demonstrate that pump regulation through the R domain is crucial for land plant fitness and by controlling growth and nutrient uptake might have been necessary already for the successful water-to-land transition of plants.
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Arabidopsis , Bombas de Protones , Bombas de Protones/genética , Transporte Biológico , Membrana Celular , Protones , Agua , Arabidopsis/genética , Adenosina TrifosfatasasRESUMEN
Root hairs are tubular outgrowths of epidermal cells that increase the root surface area and thereby make the root more efficient at absorbing water and nutrients. Their expansion is limited to the root hair apex, where growth is reported to take place in a pulsating manner. These growth pulses coincide with oscillations of the apoplastic and cytosolic pH in a similar way as has been reported for pollen tubes. Likewise, the concentrations of apoplastic reactive oxygen species (ROS) and cytoplasmic Ca2+ oscillate with the same periodicity as growth. Whereas ROS appear to control cell wall extensibility and opening of Ca2+ channels, the role of protons as a growth signal in root hairs is less clear and may differ from that in pollen tubes where plasma membrane H+-ATPases have been shown to sustain growth. In this review, we outline our current understanding of how pH contributes to root hair development.
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Perennial grain crops could make a valuable addition to sustainable agriculture, potentially even as an alternative to their annual counterparts. The ability of perennials to grow year after year significantly reduces the number of agricultural inputs required, in terms of both planting and weed control, while reduced tillage improves soil health and on-farm biodiversity. Presently, perennial grain crops are not grown at large scale, mainly due to their early stages of domestication and current low yields. Narrowing the yield gap between perennial and annual grain crops will depend on characterizing differences in their life cycles, resource allocation, and reproductive strategies and understanding the trade-offs between annualism, perennialism, and yield. The genetic and biochemical pathways controlling plant growth, physiology, and senescence should be analyzed in perennial crop plants. This information could then be used to facilitate tailored genetic improvement of selected perennial grain crops to improve agronomic traits and enhance yield, while maintaining the benefits associated with perennialism.
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Halophytes tolerate high salinity levels that would kill conventional crops. Understanding salt tolerance mechanisms will provide clues for breeding salt-tolerant plants. Many halophytes, such as quinoa (Chenopodium quinoa), are covered by a layer of epidermal bladder cells (EBCs) that are thought to mediate salt tolerance by serving as salt dumps. We isolated an epidermal bladder cell-free (ebcf) quinoa mutant that completely lacked EBCs and was mutated in REBC and REBC-like1. This mutant showed no loss of salt stress tolerance. When wild-type quinoa plants were exposed to saline soil, EBCs accumulated potassium (K+ ) as the major cation, in quantities far exceeding those of sodium (Na+ ). Emerging leaves densely packed with EBCs had the lowest Na+ content, whereas old leaves with deflated EBCs served as Na+ sinks. When the leaves expanded, K+ was recycled from EBCs, resulting in turgor loss that led to a progressive deflation of EBCs. Our findings suggest that EBCs in young leaves serve as a K+ -powered hydrodynamic system that functions as a water sink for solute storage. Sodium ions accumulate within old leaves that subsequently wilt and are shed. This mechanism improves the survival of quinoa under high salinity conditions.
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Chenopodium quinoa , Plantas Tolerantes a la Sal , Plantas Tolerantes a la Sal/genética , Tolerancia a la Sal/genética , Chenopodium quinoa/genética , Vejiga Urinaria , Fitomejoramiento , Salinidad , Sodio , Potasio , Iones , Suelo , AguaRESUMEN
Sustainable agriculture in the future will depend on crops that are tolerant to biotic and abiotic stresses, require minimal input of water and nutrients and can be cultivated with a minimal carbon footprint. Wild plants that fulfill these requirements abound in nature but are typically low yielding. Thus, replacing current high-yielding crops with less productive but resilient species will require the intractable trade-off of increasing land area under cultivation to produce the same yield. Cultivating more land reduces natural resources, reduces biodiversity and increases our carbon footprint. Sustainable intensification can be achieved by increasing the yield of underutilized or wild plant species that are already resilient, but achieving this goal by conventional breeding programs may be a long-term prospect. De novo domestication of orphan or crop wild relatives using mutagenesis is an alternative and fast approach to achieve resilient crops with high yields. With new precise molecular techniques, it should be possible to reach economically sustainable yields in a much shorter period of time than ever before in the history of agriculture.
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Domesticación , Fitomejoramiento , Productos Agrícolas/genética , Agricultura , BiodiversidadRESUMEN
The Mildew Resistance Locus O (MLO) promotes both symbiosis and biotrophic interactions with fungi. MLO disruption results in powdery mildew resistance but is associated with growth defects. New research by Li et al. demonstrates that they can be rescued by ectopic activation of a vacuolar hexose transporter.