CRISPR/Cas-mediated plant genome editing: outstanding challenges a decade after implementation.

The discovery of the CRISPR/Cas genome-editing system has revolutionized our understanding of the plant genome. CRISPR/Cas has been used for over a decade to modify plant genomes for the study of specific genes and biosynthetic pathways as well as to speed up breeding in many plant species, including both model and non-model crops. Although the CRISPR/Cas system is very efficient for genome editing, many bottlenecks and challenges slow down further improvement and applications. In this review we discuss the challenges that can occur during tissue culture, transformation, regeneration, and mutant detection. We also review the opportunities provided by new CRISPR platforms and specific applications related to gene regulation, abiotic and biotic stress response improvement, and de novo domestication of plants.

SabaTracheid 1.0: A Novel Program for Quantitative Analysis of Conifer Wood Anatomy - A Demonstration on African Juniper From the Blue Nile Basin.

Knowledge about past climates, especially at a seasonal time scale, is important as it allows informed decisions to be made to mitigate future climate change. However, globally, and especially in semi-arid Tropics, instrumental climatic data are scarce. A dendroclimatic approach may fill this gap, but tropical dendrochronological data are rare and do not yet provide fine resolution intra-annual information about past climates. Unlike in the Tropics, in the Mediterranean, temperate, alpine, and arctic regions, dendroanatomy and quantitative wood anatomy (QWA) are progressing fast attaining an intra-annual resolution, which allows a better understanding of seasonal climate dynamics and climate-growth relationships. The existing dendroanatomical and QWA methods aren't suitable for tropical trees because they do not consider the high variation in tree ring width and the frequent occurrence of micro-rings containing only a few tracheids per radial file. The available tracheid analysis programs generally fail to provide multiple sectors for micro-rings and they are unable to compute most of the useful dendroanatomical parameters at fine temporal resolutions. Here, we present a program (SabaTracheid) that addresses the three main standard tasks that are necessary for QWA and dendroanatomy before running a climate analysis: (1) tracheidogram standardization, (2) sectoring, and (3) computing QWA and dendroanatomical variables. SabaTracheid is demonstrated on African Juniper (Juniperus procera Hochst. ex Endl), but it is potentially able to provide fine-resolution QWA and dendroanatomic data that could be used for dendroanatomical studies in all regions of the world. SabaTracheid is a freeware that quickly and accurately standardizes tracheidograms, divides tree rings into multiple regular sectors, computes useful dendroanatomic and QWA variables for the whole tree rings, early- and latewood portions, and each sector separately. This program is particularly adapted to deal with high inter-annual growth variations observed in tropical trees so that it assures the provision of complete sectoral QWA and dendroanatomical data for micro-rings as well. We demonstrate SabaTracheid using a dataset of 30 Juniperus procera tree rings from the Blue Nile basin, in Ethiopia. SabaTracheid's ability to provide fine resolution QWA and dendroanatomic data will help the discipline develop in tropical as well as in the Mediterranean and temperate regions.

Identification and characterization of the Ipomoea nil RelA/SpoT Homologs (InRSHs) and potential directions of their transcriptional regulation.

Although the stringent response has been known for more than half a century and has been well studied in bacteria, only the research of the past 19 years revealed that the homologous mechanism is conserved in plants. The plant RelA/SpoT Homolog (RSH) genes have been identified and characterized in a limited number of plant species, whereas products of their catalytic activities, (p)ppGpp (alarmones), have been shown to accumulate mainly in chloroplasts. Here, we identified full-length sequences of the Ipomoea nil RSH genes (InRSH1, InRSH2 and InCRSH), determined their copy number in the I. nil genome as well as the structural conservancy between InRSHs and their Arabidopsis and rice orthologs. We showed that InRSHs are differentially expressed in I. nil organ tissues and that only InRSH2 is upregulated in response to salt, osmotic and drought stress. Our results of the E. coli relA/spoT mutant complementation test suggest that InRSH1 is likely a (p)ppGpp hydrolase, InCRSH - synthetase and InRSH2 shows both activities. Finally, we referred our results to the recently published I. nil genomic and proteomic data and uncovered the complexity of the I. nil RSH family as well as potential ways of the InRSH transcriptional regulation.

Within and beyond the stringent response-RSH and (p)ppGpp in plants.

MAIN CONCLUSION: Plant RSH proteins are able to synthetize and/or hydrolyze unusual nucleotides called (p)ppGpp or alarmones. These molecules regulate nuclear and chloroplast transcription, chloroplast translation and plant development and stress response. Homologs of bacterial RelA/SpoT proteins, designated RSH, and products of their activity, (p)ppGpp-guanosine tetra-and pentaphosphates, have been found in algae and higher plants. (p)ppGpp were first identified in bacteria as the effectors of the stringent response, a mechanism that orchestrates pleiotropic adaptations to nutritional deprivation and various stress conditions. (p)ppGpp accumulation in bacteria decreases transcription-with exception to genes that help to withstand or overcome current stressful situations, which are upregulated-and translation as well as DNA replication and eventually reduces metabolism and growth but promotes adaptive responses. In plants, RSH are nuclei-encoded and function in chloroplasts, where alarmones are produced and decrease transcription, translation, hormone, lipid and metabolites accumulation and affect photosynthetic efficiency and eventually plant growth and development. During senescence, alarmones coordinate nutrient remobilization and relocation from vegetative tissues into seeds. Despite the high conservancy of RSH protein domains among bacteria and plants as well as the bacterial origin of plant chloroplasts, in plants, unlike in bacteria, (p)ppGpp promote chloroplast DNA replication and division. Next, (p)ppGpp may also perform their functions in cytoplasm, where they would promote plant growth inhibition. Furthermore, (p)ppGpp accumulation also affects nuclear gene expression, i.a., decreases the level of Arabidopsis defense gene transcripts, and promotes plants susceptibility towards Turnip mosaic virus. In this review, we summarize recent findings that show the importance of RSH and (p)ppGpp in plant growth and development, and open an area of research aiming to understand the function of plant RSH in response to stress.

Protein phosphatase AP2C1 negatively regulates basal resistance and defense responses to Pseudomonas syringae.

Mitogen-activated protein kinases (MAPKs) mediate plant immune responses to pathogenic bacteria. However, less is known about the cell autonomous negative regulatory mechanism controlling basal plant immunity. We report the biological role of Arabidopsis thaliana MAPK phosphatase AP2C1 as a negative regulator of plant basal resistance and defense responses to Pseudomonas syringae. AP2C2, a closely related MAPK phosphatase, also negatively controls plant resistance. Loss of AP2C1 leads to enhanced pathogen-induced MAPK activities, increased callose deposition in response to pathogen-associated molecular patterns or to P. syringae pv. tomato (Pto) DC3000, and enhanced resistance to bacterial infection with Pto. We also reveal the impact of AP2C1 on the global transcriptional reprogramming of transcription factors during Pto infection. Importantly, ap2c1 plants show salicylic acid-independent transcriptional reprogramming of several defense genes and enhanced ethylene production in response to Pto. This study pinpoints the specificity of MAPK regulation by the different MAPK phosphatases AP2C1 and MKP1, which control the same MAPK substrates, nevertheless leading to different downstream events. We suggest that precise and specific control of defined MAPKs by MAPK phosphatases during plant challenge with pathogenic bacteria can strongly influence plant resistance.

MAPK phosphatase AP2C3 induces ectopic proliferation of epidermal cells leading to stomata development in Arabidopsis.

In plant post-embryonic epidermis mitogen-activated protein kinase (MAPK) signaling promotes differentiation of pavement cells and inhibits initiation of stomata. Stomata are cells specialized to modulate gas exchange and water loss. Arabidopsis MAPKs MPK3 and MPK6 are at the core of the signaling cascade; however, it is not well understood how the activity of these pleiotropic MAPKs is constrained spatially so that pavement cell differentiation is promoted only outside the stomata lineage. Here we identified a PP2C-type phosphatase termed AP2C3 (Arabidopsis protein phosphatase 2C) that is expressed distinctively during stomata development as well as interacts and inactivates MPK3, MPK4 and MPK6. AP2C3 co-localizes with MAPKs within the nucleus and this localization depends on its N-terminal extension. We show that other closely related phosphatases AP2C2 and AP2C4 are also MAPK phosphatases acting on MPK6, but have a distinct expression pattern from AP2C3. In accordance with this, only AP2C3 ectopic expression is able to stimulate cell proliferation leading to excess stomata development. This function of AP2C3 relies on the domains required for MAPK docking and intracellular localization. Concomitantly, the constitutive and inducible AP2C3 expression deregulates E2F-RB pathway, promotes the abundance and activity of CDKA, as well as changes of CDKB1;1 forms. We suggest that AP2C3 downregulates the MAPK signaling activity to help maintain the balance between differentiation of stomata and pavement cells.