Getting to the roots of N, P, and K uptake.

The soil contributes to the main pool of essential mineral nutrients for plants. These mineral nutrients are critical elements for the building blocks of plant biomolecules, play fundamental roles in cell processes, and act in various enzymatic reactions. The roots are the main entry point for mineral nutrients used within the plant to grow, develop, and produce seeds. In this regard, a suite of plant nutrient transport systems, sensors, and signaling proteins function in acquiring mineral nutrients through the roots. Mineral nutrients from chemical fertilizers, composed mainly of nitrogen, phosphorus, and potassium (NPK), are added to agricultural land to maximize crop yields, worldwide. However, improving nutrient uptake and use within crops is critical for economically and environmentally sustainable agriculture. Therefore, we review the molecular basis for N, P, and K nutrient uptake into the roots. Remarkably, plants are responsive to heterogeneous nutrient distribution and align root growth and nutrient uptake with nutrient-rich patches. We highlight the relationship between nutrient distribution in the growth environment and root system architecture. We discuss the exchange of information between the root and shoot systems through the xylem and phloem, which coordinates nutrient uptake with photosynthesis. The size and structure of the root system, along with the abundance and activity of nutrient transporters, largely determine the nutrient acquisition rate. Lastly, we discuss connections between N, P, and K uptake and signaling.

Advanced domestication: harnessing the precision of gene editing in crop breeding.

Human population growth has increased the demand for food crops, animal feed, biofuel and biomaterials, all the while climate change is impacting environmental growth conditions. There is an urgent need to develop crop varieties which tolerate adverse growth conditions while requiring fewer inputs. Plant breeding is critical to global food security and, while it has benefited from modern technologies, it remains constrained by a lack of valuable genetic diversity, linkage drag, and an effective way to combine multiple favourable alleles for complex traits. CRISPR/Cas technology has transformed genome editing across biological systems and promises to transform agriculture with its high precision, ease of design, multiplexing ability and low cost. We discuss the integration of CRISPR/Cas-based gene editing into crop breeding to advance domestication and refine inbred crop varieties for various applications and growth environments. We highlight the use of CRISPR/Cas-based gene editing to fix desirable allelic variants, generate novel alleles, break deleterious genetic linkages, support pre-breeding and for introgression of favourable loci into elite lines.

CRISPR/Cas9 editing of three CRUCIFERIN C homoeologues alters the seed protein profile in Camelina sativa.

BACKGROUND: The oilseed Camelina sativa is grown for a range of applications, including for biofuel, biolubricants, and as a source of omega-3 fatty acids for the aquaculture feed industry. The seed meal co-product is used as a source of protein for animal feed; however, the low value of the meal hinders profitability and more widespread application of camelina. The nutritional quality of the seed meal is largely determined by the abundance of specific seed storage proteins and their amino acid composition. Manipulation of seed storage proteins has been shown to be an effective means for either adjustment of nutritional content of seeds or for enhancing accumulation of high-value recombinant proteins in seeds. RESULTS: CRISPR/Cas9 gene editing technology was used to generate deletions in the first exon of the three homoeologous genes encoding the seed storage protein CRUCIFERIN C (CsCRUC), creating an identical premature stop-codon in each and resulting in a CsCRUC knockout line. The mutant alleles were detected by applying a droplet digital PCR drop-off assay. The quantitative nature of this technique is particularly valuable when applied to polyploid species because it can accurately determine the number of mutated alleles in a gene family. Loss of CRUC protein did not alter total seed protein content; however, the abundance of other cruciferin isoforms and other seed storage proteins was altered. Consequently, seed amino acid content was significantly changed with an increase in the proportion of alanine, cysteine and proline, and decrease of isoleucine, tyrosine and valine. CsCRUC knockout seeds did not have changed total oil content, but the fatty acid profile was significantly altered with increased relative abundance of all saturated fatty acids. CONCLUSIONS: This study demonstrates the plasticity of the camelina seed proteome and establishes a CRUC-devoid line, providing a framework for modifying camelina seed protein composition. The results also illustrate a possible link between the composition of the seed proteome and fatty acid profile.

The Kinase Activity of Calcineurin B-like Interacting Protein Kinase 26 (CIPK26) Influences Its Own Stability and that of the ABA-regulated Ubiquitin Ligase, Keep on Going (KEG).

The Really Interesting New Gene (RING)-type E3 ligase, Keep on Going (KEG) plays a critical role in Arabidopsis growth after germination and the connections between KEG and hormone signaling pathways are expanding. With regards to abscisic acid (ABA) signaling, KEG targets ABA-responsive transcription factors abscisic acid insensitive 5, ABF1 and ABF3 for ubiquitination and subsequent degradation through the 26S proteasome. Regulation of E3 ligases through self-ubiquitination is common to RING-type E3 ligases and ABA promotes KEG self-ubiquitination and degradation. ABA-mediated degradation of KEG is phosphorylation-dependent; however, upstream signaling proteins that may regulate KEG stability have not been characterized. In this report, we show that CBL-Interacting Protein Kinase (CIPK) 26 can phosphorylate KEG in vitro. Using both in vitro and in planta degradation assays we provide evidence which suggests that the kinase activity of CIPK26 promotes the degradation of KEG. Furthermore, we found that the kinase activity of CIPK26 also influences its own stability; a constitutively active version is more stable than a wild type or a kinase dead version. Our results suggest a reciprocal regulation model wherein an activated and stable CIPK26 phosphorylates KEG to promote degradation of the E3.

Arabidopsis CIPK26 interacts with KEG, components of the ABA signalling network and is degraded by the ubiquitin-proteasome system.

The RING-type E3 ligase, Keep on Going (KEG), is required for early seedling establishment in Arabidopsis thaliana. Post-germination, KEG negatively regulates abscisic acid (ABA) signalling by targeting Abscisic Acid Insensitive 5 (ABI5) for ubiquitination and subsequent degradation. Previous reports suggest that the role of KEG during early seedling development is not limited to regulation of ABI5 abundance. Using a yeast two-hybrid screen, this study identified Calcineurin B-like Interacting Protein Kinase (CIPK) 26 as a KEG-interacting protein. In vitro pull-down and in planta bimolecular fluorescence complementation assays confirmed the interactions between CIPK26 and KEG. In planta experiments demonstrated that CIPK26 was ubiquitinated and degraded via the 26S proteasome. It was also found that turnover of CIPK26 was increased when KEG protein levels were elevated, suggesting that the RING-type E3 ligase is involved in targeting CIPK26 for degradation. CIPK26 was found to interact with the ABA signalling components ABI1, ABI2, and ABI5. In addition, CIPK26 was capable of phosphorylating ABI5 in vitro. Consistent with a role in ABA signalling, overexpression of CIPK26 increased the sensitivity of germinating seeds to the inhibitory effects of ABA. The data presented in this report suggest that KEG mediates the proteasomal degradation of CIPK26 and that CIPK26 is part of the ABA signalling network.

Regulation of ethylene biosynthesis through protein degradation.

The function of hormones during plant growth, development and response to environmental stresses relies heavily upon the actions of the ubiquitin proteasome system (UPS), which selectively degrades numerous proteins. Synthesis of ethylene, a growth and stress hormone, is regulated in part by the ubiquitin-dependent degradation of the rate-limiting enzymatic protein aminocyclopropane-1-carboxylic acid synthase (ACS). Regulation of ACS protein stability, and therefore ethylene production, is mediated by non-catalytic sequences within the C-terminal extension of many ACS proteins. In this review we provide a brief overview of the E3 ligases that target ACS proteins for degradation and discuss how post-translational modification of the C-terminal extensions influence protein stability.

The Arabidopsis RING-type E3 ligase XBAT32 mediates the proteasomal degradation of the ethylene biosynthetic enzyme, 1-aminocyclopropane-1-carboxylate synthase 7.

E3 ubiquitin ligases select specific proteins for ubiquitin conjugation, and the modified proteins are commonly degraded through the 26S proteasome. XBAT32 is a RING-type E3 ligase involved in maintaining appropriate levels of ethylene. Previous work has suggested that XBAT32 modulates ethylene production by ubiquitinating two ethylene biosynthesis enzymes, ACS4 (type-II isoform) and ACS7 (type-III isoform). In Arabidopsis, conserved sequences within the C-terminal tail of type-I and -II 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS) isoforms influence ubiquitin-dependent proteolysis. ACS7, the sole Arabidopsis type-III ACS, contains a truncated C-terminal tail that lacks all known regulatory sequences, which suggests that this isoform may not be subject to ubiquitin-mediated proteasomal degradation. Here we demonstrate in planta that ACS7 is turned over in a 26S proteasome-dependent manner and that degradation of ACS7 requires the E3 ligase XBAT32. Furthermore, the ethylene-related phenotypes that result from overexpression of ACS7 in wild-type plants are greatly exaggerated in xbat32-1, suggesting that XBAT32 is required to attenuate the effect of overexpression of ACS7. This observation is consistent with a role for XBAT32 in the ubiquitin-mediated degradation of ACS7. The dark-grown phenotype of xbat32-1 seedlings overexpressing ACS7 can be effectively rescued by aminoethoxyvinylglycine, an inhibitor of ACS activity. The degradation rate of ACS4 is also significantly slower in the absence of XBAT32, further implicating XBAT32 in the ubiquitin-mediated degradation of ACS4. Altogether, these results demonstrate that XBAT32 targets ethylene biosynthetic enzymes for proteasomal degradation to maintain appropriate levels of hormone production.

Abiotic stress tolerance mediated by protein ubiquitination.

Plant growth and development is largely influenced by ubiquitin-mediated regulation of protein stability. Specificity of the ubiquitination pathway is controlled mainly by the substrate-recruiting E3 ubiquitin ligases, and consequently, E3 ligases control numerous cellular processes. Recent evidence that ubiquitination plays a critical role in regulating plant responses to abiotic stresses has launched intensive efforts to identify E3 ligases that mediate plant tolerance of adverse environmental conditions. Most stress-related E3 ligases identified to date facilitate responses to environmental stimuli by modulating the abundance of key downstream stress-responsive transcription factors. In this review, the regulatory roles of ubiquitin during the plant's response to abiotic stress are summarized and highlighted.

Specific aminopeptidases of excised human nasal epithelium and primary culture: a comparison of functional characteristics and gene transcripts expression.

OBJECTIVES: To investigate whether growing human nasal epithelium as primary cultures alters aminopeptidase B (APB), aminopeptidase N (APN) and dipeptidyldipeptidase (DPPIV) metabolic characteristics, and mRNA gene transcript expression. METHODS: The formation of 7-amino-methyl coumarin from specific substrates for APN (L-alanine-4-methyl-coumaryl-7-amide, APB (L-arginine-4-methyl-coumaryl-7-amide) and DPPIV (glycyl-L-proline-4-methyl-coumaryl-7-amide) was used to estimate the KM, Vmax and the effect of aminopeptidases inhibitors on the enzymes. Polymerase chain reaction was used to investigate gene expression. KEY FINDINGS: Results of this study showed that: (1) both the excised tissues and primary cultures of human nasal epithelium expressed APN, APB and DPPIV activity; (2) the KM of APB, APN and DPPIV was not significantly different in cell and tissue homogenates; (3) except for APN, the Vmax was not significantly different in the two metabolism models; (4) there was no statistically significant difference in the behaviours of APB, APN and DPPIV in response to inhibition by puromycin and bestatin in the two models; (5) the mRNA transcripts that encode APB, APN and DPPIV were expressed in both cell culture and tissue homogenate. CONCLUSIONS: Based on the results of this study, it may be concluded that nasal primary culture system is suitable for investigating peptide and protein metabolism and enzymatic stability in human nasal epithelium. Except for APN, the tissue culture conditions did not significantly alter the functional and molecular expression of the aminopeptidases.