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In the last years, tremendous progress has been made in the development of CRISPR/Cas-mediated genome editing tools. A number of natural CRISPR/Cas nuclease variants have been characterized. Engineered Cas proteins have been developed to minimize PAM restrictions, off-side effects and temperature sensitivity. Both kinds of enzymes have, by now, been applied widely and efficiently in many plant species to generate either single or multiple mutations at the desired loci by multiplexing. In addition to DSB-induced mutagenesis, specifically designed CRISPR/Cas systems allow more precise gene editing, resulting not only in random mutations but also in predefined changes. Applications in plants include gene targeting by homologous recombination, base editing and, more recently, prime editing. We will evaluate these different technologies for their prospects and practical applicability in plants. In addition, we will discuss a novel application of the Cas9 nuclease in plants, enabling the induction of heritable chromosomal rearrangements, such as inversions and translocations. This technique will make it possible to change genetic linkages in a programmed way and add another level of genome engineering to the toolbox of plant breeding. Also, strategies for tissue culture free genome editing were developed, which might be helpful to overcome the transformation bottlenecks in many crops. All in all, the recent advances of CRISPR/Cas technology will help agriculture to address the challenges of the twenty-first century related to global warming, pollution and the resulting food shortage.

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Nicotiana tabacum is a non-food herb that has the potential to be utilized as bio-factory for generating medicines, vaccines or valuable small metabolites. To achieve these goals, the improvement of genetic tools for pre-designed genome modifications is indispensable. The development of CRISPR/Cas nucleases allows the induction of site-specific double-strand breaks to enhance homologous recombination-mediated gene targeting (GT). However, the efficiency of GT is still a challenging obstacle for many crops including tobacco. Recently, studies in several plant species indicated that by replacing SpCas9 with other CRISPR/Cas-based nucleases, GT efficiencies might be enhanced considerably. Therefore, we tested SaCas9 as well as a temperature-insensitive version of LbCas12a (ttLbCas12a) for targeting the tobacco SuRB gene. At the same time, we also optimized the protocol for Agrobacterium-mediated tobacco transformation and tissue culture. In this way, we could improve GT efficiencies to up to a third of the inoculated cotyledons when using ttLbCas12a, which outperformed SaCas9 considerably. In addition, we could show that the conversion tract length of the GT reaction can be up to 606 bp long and in the majority of cases, it is longer than 250 bp. We obtained multiple heritable GT events, mostly heterozygous, but also biallelic GT events and some without T-DNA integration. Thus, we were not only able to obtain CRISPR/Cas-based heritable GT events in allotetraploid Nicotiana tabacum for the first time, but our results also indicate that ttLbCas12a might be a superior alternative for gene editing and GT in tobacco as well as in other crops.

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KEY MESSAGE: We summarize recent progress of CRISPR/Cas9-mediated gene targeting in plants, provide recommendations for designing gene-targeting vectors and highlight the potential of new technologies applicable to plants. Gene targeting (GT) is a tool of urgent need for plant biotechnology and breeding. It is based on homologous recombination that is able to precisely introduce desired modifications within a target locus. However, its low efficiency in higher plants is a major barrier for its application. Using site-specific nucleases, such as the recent CRISPR/Cas system, GT has become applicable in plants, via the induction of double-strand breaks, although still at a too low efficiency for most practical applications in crops. Recently, a variety of promising new improvements regarding the efficiency of GT has been reported by several groups. It turns out that GT can be enhanced by cell-type-specific expression of Cas nucleases, by the use of self-amplified GT-vector DNA or by manipulation of DNA repair pathways. Here, we highlight the most recent progress of GT in plants. Moreover, we provide suggestions on how to use the technology efficiently, based on the mechanisms of DNA repair, and highlight several of the newest GT strategies in yeast or mammals that are potentially applicable to plants. Using the full potential of GT technology will definitely help us pave the way in enhancing crop yields and food safety for an ecologically friendly agriculture.

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Vacuoles play a fundamental role in storage and remobilization of various nutrients, including phosphorus (P), an essential element for cell growth and development. Cells acquire P primarily in the form of inorganic orthophosphate (Pi). However, the form of P stored in vacuoles varies by organism and tissue. Algae and yeast store polyphosphates (polyPs), whereas plants store Pi and inositol phosphates (InsPs) in vegetative tissues and seeds, respectively. In this review, we summarize how vacuolar P molecules are stored and reallocated and how these processes are regulated and co-ordinated. The roles of SYG1/PHO81/XPR1 (SPX)-domain-containing membrane proteins in allocating vacuolar P are outlined. We also highlight the importance of vacuolar P in buffering the cytoplasmic Pi concentration to maintain cellular homeostasis when the external P supply fluctuates, and present additional roles for vacuolar polyP and InsP besides being a P reserve. Furthermore, we discuss the possibility of alternative pathways to recycle Pi from other P metabolites in vacuoles. Finally, future perspectives for researching this topic and its potential application in agriculture are proposed.

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Plant vacuoles serve as the primary intracellular compartments for inorganic phosphate (Pi) storage. Passage of Pi across vacuolar membranes plays a critical role in buffering the cytoplasmic Pi level against fluctuations of external Pi and metabolic activities. Here we demonstrate that the SPX-MFS proteins, designated as PHOSPHATE TRANSPORTER 5 family (PHT5), also named Vacuolar Phosphate Transporter (VPT), function as vacuolar Pi transporters. Based on (31)P-magnetic resonance spectroscopy analysis, Arabidopsis pht5;1 loss-of-function mutants accumulate less Pi and exhibit a lower vacuolar-to-cytoplasmic Pi ratio than controls. Conversely, overexpression of PHT5 leads to massive Pi sequestration into vacuoles and altered regulation of Pi starvation-responsive genes. Furthermore, we show that heterologous expression of the rice homologue OsSPX-MFS1 mediates Pi influx to yeast vacuoles. Our findings show that a group of Pi transporters in vacuolar membranes regulate cytoplasmic Pi homeostasis and are required for fitness and plant growth.

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Phosphate (Pi), which is indispensable for the structural and metabolic needs of plants, is acquired and translocated by Pi transporters. Deciphering the regulatory network of Pi signaling and homeostasis that involves the control of Pi transporters trafficking to, and their activity at, the plasma membrane provides insight into how plants adapt to environmental changes in Pi availability. Here, we review recent studies that revealed the involvement of microRNA399-PHOSPHATE 2 (PHO2) and microR827-NITROGEN LIMITATION ADAPTATION (NLA) modules in mediating the ubiquitination and degradation of PHOSPHATE TRANSPORTER 1 (PHT1) and/or PHOSPHATE 1 (PHO1). These discoveries show that miRNAs are an effective way for plants to monitor the turnover of Pi transporters in the membrane system by modulating the functioning of the membrane-associated ubiquitin machinery.

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Phosphate (Pi) is an essential nutrient for plants but is normally fixed in soil, which limits plant growth and reproduction. In response to low availability of Pi, shoots and roots react differently but cooperatively to improve Pi acquisition from the rhizosphere and adjust Pi distribution and metabolism within plants. Shoot and root responses are coordinated by the trafficking of various kinds of systemic signals through the vasculature. Mutual communication between different tissues is necessary to integrate the environmental stimuli with the internal cues at the whole-plant level. Different approaches have been used to monitor or manipulate components in the vascular stream to reveal several candidates of systemic signals from roots or shoots, including photosynthates, phytohormones, microRNAs, and Pi. In addition, the downstream signalling pathways mediated by these signals have been discovered. The crosstalk among different signalling pathways has been revealed, showing the complexity of the Pi signalling network. In this review, we summarize the approaches used for studying systemic signalling and discuss recent progress and challenges in investigating the systemic signalling pathway that integrates Pi starvation responses to maintain Pi at physiological concentrations. Knowledge gained from this study may help improve the phosphorus use efficiency of crops.

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MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/phosphate2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ (for isobaric tags for relative and absolute quantitation)- based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in phosphate transporter1 (PHT1) family and phosphate transporter traffic facilitator1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research.

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Members of the Arabidopsis thaliana phosphate transporter1 (PHT1) family are key players in acquisition of Pi from the rhizosphere, and their regulation is indispensable for the maintenance of cellular Pi homeostasis. Here, we reveal posttranslational regulation of Pi transport through modulation of degradation of PHT1 proteins by the RING-type ubiquitin E3 ligase, nitrogen limitation adaptation (NLA). Loss of function of NLA caused high Pi accumulation resulting from increases in the levels of several PHT1s at the protein rather than the transcript level. Evidence of decreased endocytosis and ubiquitination of PHT1s in nla mutants and interaction between NLA and PHT1s in the plasma membranes suggests that NLA directs the ubiquitination of plasma membrane-localized PHT1s, which triggers clathrin-dependent endocytosis followed by endosomal sorting to vacuoles. Furthermore, different subcellular localization of NLA and phosphate2 (pho2; a ubiquitin E2 conjugase) and the synergistic effect of the accumulation of PHT1s and Pi in nla pho2 mutants suggest that they function independently but cooperatively to regulate PHT1 protein amounts. Intriguingly, NLA and PHO2 are the targets of two Pi starvation-induced microRNAs, miR827 and miR399, respectively. Therefore, our findings uncover modulation of Pi transport activity in response to Pi availability through the integration of a microRNA-mediated posttranscriptional pathway and a ubiquitin-mediated posttranslational regulatory pathway.

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The Arabidopsis thaliana pho2 mutant, which is defective in a ubiquitin-conjugating E2 enzyme, displays inorganic phosphate (Pi) toxicity as a result of enhanced uptake and root-to-shoot translocation of Pi. To elucidate downstream components of the PHO2-dependent regulatory pathway, we identified two pho2 suppressors as carrying missense mutations in PHO1, which has been implicated in Pi loading to the xylem. In support of the genetic interaction between PHO1 and PHO2, we found that the protein level of PHO1 is increased in pho2, whereas such accumulation is ameliorated in both pho2 suppressors. Results from cycloheximide and endosomal Cys protease inhibitor E-64d treatments further suggest that PHO1 degradation is PHO2 dependent and involves multivesicular body-mediated vacuolar proteolysis. Using the transient expression system of tobacco (Nicotiana tabacum) leaves, we demonstrated that PHO1 and PHO2 are partially colocalized and physically interact in the endomembranes, where the ubiquitin conjugase activity of PHO2 is required for PHO1 degradation. In addition, reduced PHO1 expression caused by PHO1 mutations impede Pi uptake, indicating a functional association between xylem loading and acquisition of Pi. Together, our findings uncover a pivotal molecular mechanism by which PHO2 modulates the degradation of PHO1 in the endomembranes to maintain Pi homeostasis in plants.

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