Single Mutants Research Articles

Comprehensive analysis of stress-activated protein kinase genes (OsSAPKs) in rice flowering time.

The rice SnRK2 members SAPK4, SAPK5, SAPK7 and SAPK10 are positive regulators involved in the regulation of rice flowering, while other single mutants exhibited no effect on rice flowering. The rice SnRK2 family, comprising 10 members known as SAPK (SnRK2-Associated Protein Kinase), is pivotal in the abscisic acid (ABA) pathway and crucial for various biological processes, such as drought resistance and salt tolerance. Additionally, these members have been implicated in the regulation of rice heading date, a key trait influencing planting area and yield. In this study, we utilized gene editing technology to create mutants in the Songjing 2 (SJ2) background, enabling a comprehensive analyze the role of each SAPK member in rice flowering. We found that SAPK1, SAPK2, and SAPK3 may not directly participate in the regulatory network of rice heading date, while SAPK4, SAPK5, and SAPK7 play positive roles in rice flowering regulation. Notably, polygene deletion resulted in an additive effect on delaying flowering. Our findings corroborate the previous studies indicating the positive regulatory role of SAPK10 in rice flowering, as evidenced by delayed flowering observed in sapk9/10 double mutants. Moving forward, our future research will focus on analyzing the molecular mechanisms underlying SAPKs involvement in rice flowering regulation, aiming to enhance our understanding of the rice heading date relationship network and lay a theoretical foundation for breeding efforts to alter rice ripening dates.

Sub-microscopic Plasmodium falciparum infections and multiple drug resistant single nucleotide polymorphic alleles in pregnant women from southwestern Nigeria

ObjectivesThe study evaluated sub-microscopic malaria infections in pregnancy using two malaria Rapid Diagnostic Tests (mRDTs), microscopy and RT-PCR and characterized Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and Plasmodium falciparum dihydropteroate synthase (Pfdhps) drug resistant markers in positive samples.MethodsThis was a cross sectional survey of 121 pregnant women. Participants were finger pricked, blood drops were collected for rapid diagnosis with P. falciparum histidine-rich protein 11 rapid diagnostic test kit and the ultra-sensitive Alere Pf malaria RDT, Blood smears for microscopy and dried blood spots on Whatman filter paper for molecular analysis were made. Real time PCR targeting the var acidic terminal sequence (varATS) gene of P. falciparum was carried out on a CFX 96 real time system thermocycler (BioRad) in discriminating malaria infections. For each run, laboratory strain of P. falciparum 3D7 and nuclease free water were used as positive and negative controls respectively. Additionally, High resolution melt analyses was employed for genotyping of the different drug resistance markers.ResultsOut of one hundred and twenty-one pregnant women sampled, the SD Bioline™ Malaria Ag P.f HRP2-based malaria rapid diagnostic test (mRDT) detected eight (0.06%) cases, the ultra-sensitive Alere™ malaria Ag P.f rapid diagnostic test mRDT had similar outcome in the same samples as detected by the HRP2-based mRDT. Microscopy and RT-PCR confirmed four out of the eight infections detected by both rapid diagnostic tests as true positive and RT-PCR further detected three false negative samples by the two mRDTs providing a sub-microscopic malaria prevalence of 3.3%. Single nucleotide polymorphism in Pfdhps gene associated with sulphadoxine resistance revealed the presence of S613 mutant genotypes in three of the seven positive isolates and isolates with mixed wild/mutant genotype at codon A613S. Furthermore, four mixed genotypes at the A581G codon were also recorded while the other Pfdhps codons (A436G, A437G and K540E) showed the presence of wild type alleles. In the Pfdhfr gene, there were mutations in 28.6%, 28.6%, and 85.7% at the I51, R59 and N108 codons respectively. Mixed wild and mutant type genotypes were also observed in 28.6% each of the N51I, and C59R codons. For the Pfcrt, two haplotypes CVMNK and CVIET were observed. The SVMNT was altogether absent. Triple mutant CVIET 1(14.3%) and triple mutant + wild genotype CVIET + CVMNK 1(14.3%) were observed. The Pfmdr1 haplotypes were single mutants YYND 1(14.3%); NFND 1(14.3%) and double mutants YFND 4(57.1%); YYDD 1(14.3%).

Cytokinin and MAX2 signaling pathways act antagonistically in drought adaptation of Arabidopsis thaliana

Understanding the mechanisms, especially those associated with phytohormones, of plant drought adaptation is crucial for sustaining agricultural production in the era of climate change. Arabidopsis histidine kinases (AHKs), an integral part of the cytokinin signaling pathway, and more axillary growth 2 (MAX2), a key component of the strigolactone and karrikin signaling pathways are reported to act as negative and postive regulators, respectively, in plant adaption to drought. However, the potential interaction between these singaling pathways in plant drought adaptation is not fully understood. To address this query, we assessed drought tolerance levels and associated phenotypic and physiological traits of the max2 single mutant, ahk2 ahk3 double mutant, ahk2 ahk3 max2 triple mutant, and wild-type (WT) Arabidopsis thaliana plants. Our findings revealed a distinct hierarchy in drought tolerance among these genotypes, as indicated by the differences in plant growth and stress survival rates. Specifically, the max2 mutant displayed the lowest drought tolerance level, followed by WT, ahk2 ahk3 max2, and ahk2 ahk3 plants. Additionally, the observed changes in leaf relative water content, leaf surface temperature, and cuticle formation were coherently aligned with the observed hierarchy of drought tolerance levels. Under drought conditions, the max2 mutant exhibited higher oxidative stress and membrane damage, as evidenced by increased levels of reactive oxygen species (ROS), malondialdehyde, and electrolyte leakage. In contrast, the ahk2 ahk3 and ahk2 ahk3 max2 mutants showed low and intermediate levels, respectively, for these parameters. The max2 mutant displayed reduced sensitivity, whereas ahk2 ahk3 and ahk2 ahk3 max2 mutants demonstrated high and intermediate sensitivities, respectively, to exogenous absicic acid (ABA) treatments. Additionally, the expression analysis of several genes associated with the investigated drought tolerance-related traits showed a positive correlation between the transcript levels and corresponding trait(s) in both mutant and WT plants under drought conditions. Our results collectively indicate the presence of an antagonistic interaction between AHK and MAX2 signaling pathways in plant drought adaptation, impacting ABA responsiveness, leaf water retention, cuticle development, and ROS homestasis. Findings of this study provide a valuable foundation for developing agricultural methods to improve plant drought resilience.

Nucleotide-binding leucine-rich repeat receptor homologs Pib and PibH8 interact and contribute to immunity in rice.

Plant nucleotide-binding leucine-rich repeat receptors (NLRs) sense pathogen effectors and activate effector-triggered immunity (ETI). Many plant NLRs form pairs with other NLRs to recognize effectors and initiate ETI. PIRICULARIA ORYZAE RESISTANCE IN BL1 (Pib), an NLR protein in rice (Oryza sativa), activates resistance by recognizing the rice blast effector AvrPib. The activation of Pib is suppressed by SH3 DOMAIN-CONTAINING PROTEIN 2 (OsSH3P2) in the absence of AvrPib. However, how Pib triggers defense responses and whether Pib pairs with another NLR are not clear. In this study, we identified Pib by map-based cloning and showed that a homolog of Pib, PIB HOMOLOGUE 8 (PibH8), interacts with Pib. Pib and PibH8 mediate resistance to the Magnaporthe oryzae isolate Guy11, a rice blast strain carrying AvrPib. Interestingly, the pib/pibh8 double mutant exhibited enhanced susceptibility to Guy11 compared to the single mutant. Furthermore, PibH8 can oligomerize through its coiled-coil (CC) domain, which also contributes to the Pib-PibH8 interaction, suggesting that Pib and PibH8 may form a complex to recognize AvrPib. OsSH3P2 inhibited the interaction of Pib and PibH8 through association with the CC domain of PibH8. Taken together, these results indicate that both Pib and PibH8 are required for rice blast resistance to M. oryzae carrying AvrPib, which is negatively regulated by OsSH3P2. This study not only identifies an NLR that functions in rice blast resistance but also reveals a possible complex immune strategy in which homologous NLR proteins may regulate the complete activation of plant immunity.

Does Acinetobacter calcoaceticus glucose dehydrogenase produce self-damaging H2O2?

The soluble glucose dehydrogenase (sGDH) from Acinetobacter calcoaceticus has been widely studied and is used, in biosensors, to detect the presence of glucose, taking advantage of its high turnover and insensitivity to molecular oxygen. This approach, however, presents two drawbacks: the enzyme has broad substrate specificity (leading to imprecise blood glucose measurements) and shows instability over time (inferior to other oxidizing glucose enzymes). We report the characterization of two sGDH mutants: the single mutant Y343F, and the double mutant D143E/Y343F. The mutants present enzyme selectivity and specificity of 1.2 (Y343F) and 5.7 (D143E/Y343F) times higher for glucose compared to that of the wild type. Crystallographic experiments, designed to characterise these mutants, surprisingly revealed that the prosthetic group PQQ (pyrroloquinoline quinone), essential for the enzymatic activity, is in a cleaved form for both wild-type and mutant structures. We provide evidence suggesting that the sGDH produces H2O2, the level of production depending on the mutation. In addition, spectroscopic experiments allowed us to follow the self-degradation of the prosthetic group and the disappearance of sGDH's glucose oxidation activity. These studies suggest that the enzyme is sensitive to its self-production of H2O2. We show that the premature aging of sGDH can be slowed down by adding catalase to consume the H2O2 produced, allowing the design of a more stable biosensor over time. Our research opens questions about the mechanism of H2O2 production and the physiological role of this activity by sGDH.

Is the A-1 Pigment in Photosystem I Part of P700? A (P700+-P700) FTIR Difference Spectroscopy Study of A-1 Mutants.

The involvement of the second pair of chlorophylls, termed A-1A and A-1B, in light-induced electron transfer in photosystem I (PSI) is currently debated. Asparagines at PsaA600 and PsaB582 are involved in coordinating the A-1B and A-1A pigments, respectively. Here we have mutated these asparagine residues to methionine in two single mutants and a double mutant in PSI from Synechocystis sp. PCC 6803, which we term NA600M, NB582M, and NA600M/NB582M mutants. (P700+-P700) FTIR difference spectra (DS) at 293 K were obtained for the wild-type and the three mutant PSI samples. The wild-type and mutant FTIR DS differ considerably. This difference indicates that the observed changes in the (P700+-P700) FTIR DS cannot be due to only the PA and PB pigments of P700. Comparison of the wild-type and mutant FTIR DS allows the assignment of different features to both A-1 pigments in the FTIR DS for wild-type PSI and assesses how these features shift upon cation formation and upon mutation. While the exact role the A-1 pigments play in the species we call P700 is unclear, we demonstrate that the vibrational modes of the A-1A and A-1B pigments are modified upon P700+ formation. Previously, we showed that the A-1 pigments contribute to P700 in green algae. In this manuscript, we demonstrate that this is also the case in cyanobacterial PSI. The nature of the mutation-induced changes in algal and cyanobacterial PSI is similar and can be considered within the same framework, suggesting a universality in the nature of P700 in different photosynthetic organisms.

The Characterization of Multifaceted PREP1 Peptides Provides Insights into Correlations between Spectroscopic and Structural Properties of Amyloid-like Assemblies.

The widespread ability of proteins and peptides to self-assemble by forming cross-β structure is one of the most significant discoveries in structural biology. Intriguingly, the cross-β association of proteins/peptides may generate intricate supramolecular architectures with uncommon spectroscopic properties. We have recently characterized self-assembled peptides extracted from the PREP1 protein that are endowed with interesting structural/spectroscopic properties. We here demonstrate that the green fluorescence emission of the peptide PREP1[117-132] (λem ~ 520 nm), can be induced by excitation with UV radiation. The associated unusually large Stokes shift (Δλ ~ 150 nm) represents, to the best of our knowledge, the first evidence of an internal resonance energy transfer in amyloid-like structures, where the blue emission of some assemblies becomes the excitation radiation for others. Moreover, the characterization of PREP1[117-132] variants provides insights into the sequence/structure and structure/spectroscopic properties relationships. Our data suggests that the green fluorescence is plausibly associated with antiparallel β-sheet states of the peptide whereas parallel β-sheet assemblies are only endowed with blue fluorescence. Notably, the different PREP1[117-132] variants also form assemblies characterized by distinct morphologies. Indeed, the parent peptide and single mutants form compact but structured aggregates whereas most of the double mutants exhibit elongated and highly extended fibers.

Evolutionary Adaptation of an RNA Bacteriophage to Repeated Freezing and Thawing Cycles.

Bacteriophage fitness is determined by factors influencing both their replication within bacteria and their ability to maintain infectivity between infections. The latter becomes particularly crucial under adverse environmental conditions or when host density is low. In such scenarios, the damage experienced by viral particles could lead to the loss of infectivity, which might be mitigated if the virus undergoes evolutionary optimization through replication. In this study, we conducted an evolution experiment involving bacteriophage Qβ, wherein it underwent 30 serial transfers, each involving a cycle of freezing and thawing followed by replication of the surviving viruses. Our findings show that Qβ was capable of enhancing its resistance to this selective pressure through various adaptive pathways that did not impair the virus replicative capacity. Notably, these adaptations predominantly involved mutations located within genes encoding capsid proteins. The adapted populations exhibited higher resistance levels than individual viruses isolated from them, and the latter surpassed those observed in single mutants generated via site-directed mutagenesis. This suggests potential interactions among mutants and mutations. In conclusion, our study highlights the significant role of extracellular selective pressures in driving the evolution of phages, influencing both the genetic composition of their populations and their phenotypic properties.

P1' specificity of the S219V/R203G mutant tobacco etch virus protease.

Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1' autoproteolytic cleavage-resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self-inactivation, but also exhibited greater stability and catalytic efficiency than the wild-type enzyme. An R203G substitution has been reported to further relax the P1' specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1' specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1' amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1' variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme-substrate interactions were analyzed in silico. The results indicate highly similar P1' preferences for both enzymes, many side-chains can be accommodated by the S1' binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.

Protein engineering of lipase A from Candida antarctica to improve esterification of tertiary alcohols.

Chiral tertiary alcohols are important organic compounds in science as well as in industry. However, their preparation in enantiomerically pure form is still a challenge due to their complex structure and steric hindrances compared with primary and secondary alcohols, so kinetic resolution could be an attractive approach. Lipase A from Candida antarctica (CAL-A) has been shown to catalyze the enantioselective esterification of various tertiary alcohols with excellent enantioselectivity but low activity. Here we report a mutagenesis study by rational design to improve CAL-A activity against tertiary alcohols. Single mutants of CAL-A were selected, expressed, immobilized and screened for esterification of the tertiary alcohol 1,2,3,4-tetrahydronaphthalene-1-ol. A double mutant V278S+S429G showed a 1.5-fold higher reaction rate than that of the wild type CAL-A, while maintaining excellent enantioselectivity.

Semirational Design Strategy To Enhance the Thermostability and Catalytic Activity of Cytochrome P450 105D7 for the Degradation of the Pharmaceutically Active Compounds: Diclofenac.

Pharmaceutically active compounds are an important category of emerging pollutants, and their biological transformation processes in the environment are crucial for understanding and evaluating the migration, transformation, and environmental fate of emerging pollutants. The cytochrome P450 105 enzyme family has been proven to play an important role in the degradation of exogenous environmental pollutants. However, its thermostability and catalytic activity still need to be improved to better adapt to complex environmental conditions. This work elucidates the key mechanisms and important residues of the degradation reaction through multiple computational strategies, establishes a mutation library, and obtains 21 single-point mutation designs. Experimental verification showed that 16 single mutants had enhanced thermostability, with the R89F and L197Y mutants showing the highest increases in thermostability at 135 and 119% relative to the wild-type enzyme, respectively. Additionally, as a result of the higher specific activity of D390Q, it was selected for combination mutagenesis, ultimately resulting in three combination mutants (R89F/L197Y, R89F/D390Q, and R89F/L197Y/D390Q) with enhanced thermostability and catalytic activity. This study provides a modification approach for constructing efficient enzyme variants through semirational design and can contribute to the development of control technologies for emerging pollutants.

Evidence for partial functional overlap of KEA and MSL transport proteins in the chloroplast inner envelope of Arabidopsis thaliana.

Arabidopsis thaliana possesses two different ion-export mechanisms in the plastid inner envelope membrane. Due to a genome duplication, the transport proteins are encoded by partly redundant loci: K+-efflux antiporter1 (KEA1) and KEA2 and mechanosensitive channel of small conductance-like2 (MSL2) and MSL3. Thus far, a functional link between these two mechanisms has not been established. Here, we show that kea1msl2 loss-of-function mutants exhibit phenotypes such as slow growth, reduced photosynthesis and changes in chloroplast morphology, several of which are distinct from either single mutants and do not resemble kea1kea2 or msl2msl3 double mutants. Our data suggest that KEA1 and MSL2 function in concert to maintain plastid ion homeostasis and osmoregulation. Their interplay is critical for proper chloroplast development, organelle function, and plant performance.

Nkd1 functions downstream of Axin2 to attenuate Wnt signaling.

Wnt signaling is a crucial developmental pathway involved in early development as well as stem cell maintenance in adults and its misregulation leads to numerous diseases. Thus, understanding the regulation of this pathway becomes vitally important. Axin2 and Nkd1 are widely utilized negative feedback regulators in Wnt signaling where Axin2 functions to destabilize cytoplasmic β-catenin, and Nkd1 functions to inhibit the nuclear localization of β-catenin. Here, we set out to further understand how Axin2 and Nkd1 regulate Wnt signaling by creating axin2gh1/gh1, nkd1gh2/gh2 single mutants and axin2gh1/gh1;nkd1gh2/gh2 double mutant zebrafish using sgRNA/Cas9. All three Wnt regulator mutants were viable and had impaired heart looping, neuromast migration defects, and behavior abnormalities in common, but there were no signs of synergy in the axin2gh1/gh1;nkd1gh2/gh2 double mutants. Further, Wnt target gene expression by qRT-PCR, and RNA-seq analysis and protein expression by mass spectrometry demonstrated that the double axin2gh1/gh1;nkd1gh2/gh2 mutant resembled the nkd1gh2/gh2 phenotype demonstrating that Nkd1 functions downstream of Axin2. In support of this, the data further demonstrates that Axin2 uniquely alters the properties of β-catenin-dependent transcription having novel readouts of Wnt activity compared to nkd1gh2/gh2 or the axin2gh1/gh1;nkd1gh2/gh2 double mutant. We also tested the sensitivity of the Wnt regulator mutants to exacerbated Wnt signaling, where the single mutants displayed characteristic heightened Wnt sensitivity, resulting in an eyeless phenotype. Surprisingly, this phenotype was rescued in the double mutant, where we speculate that cross-talk between Wnt/β-catenin and Wnt/Planar Cell Polarity pathways could lead to altered Wnt signaling in some scenarios. Collectively, the data emphasizes both the commonality and the complexity in the feedback regulation of Wnt signaling.

Mucosal adjuvanticity and mucosal booster effect of colibactin-depleted probiotic Escherichia coli membrane vesicles

ABSTRACT There is a growing interest in development of novel vaccines against respiratory tract infections, due to COVID-19 pandemic. Here, we examined mucosal adjuvanticity and the mucosal booster effect of membrane vesicles (MVs) of a novel probiotic E. coli derivative lacking both flagella and potentially carcinogenic colibactin (ΔflhDΔclbP). ΔflhDΔclbP-derived MVs showed rather strong mucosal adjuvanticity as compared to those of a single flagellar mutant strain (ΔflhD-MVs). In addition, glycoengineered ΔflhDΔclbP-MVs displaying serotype-14 pneumococcal capsular polysaccharide (CPS14+MVs) were well-characterized based on biological and physicochemical parameters. Subcutaneous (SC) and intranasal (IN) booster effects of CPS14+MVs on systemic and mucosal immunity were evaluated in mice that have already been subcutaneously prime-immunized with the same MVs. With a two-dose regimen, an IN boost (SC-IN) elicited stronger IgA responses than homologous prime-boost immunization (SC-SC). With a three-dose regimen, serum IgG levels were comparable among all tested regimens. Homologous immunization (SC-SC-SC) elicited the highest IgM responses among all regimens tested, whereas SC-SC-SC failed to elicit IgA responses in blood and saliva. Furthermore, serum IgA and salivary SIgA levels were increased with an increased number of IN doses administrated. Notably, SC-IN-IN induced not only robust IgG response, but also the highest IgA response in both serum and saliva among the groups. The present findings suggest the potential of a heterologous three-dose administration for building both systemic and mucosal immunity, e.g. an SC-IN-IN vaccine regimen could be beneficial. Another important observation was abundant packaging of colibactin in MVs, suggesting increased applicability of ΔflhDΔclbP-MVs in the context of vaccine safety.

The dynamics of colicin E9 release from Escherichia coli in native conditions.

Colicin (Col) plasmid contains colicin encoding genes arranged in an operon controlled by an SOS inducible promoter. Therefore, any external stresses to the host cell can induce the expression of the downstream genes in the Col operon, including a lysis gene. The lysis protein is involved in the extracellular release of colicin through lysis of the producer cells, which causes a decline in culture turbidity. However, it is not yet known that E. coli cells with the native pColE9-J plasmid hold the same level of cell death at the population level following a set of induced conditions. In this study, using a mitomycin C sensitivity assay along with a live dead staining method of detection, we showed that the native pColE9-J plasmid, which unusually carries an extended Col operon (ColE9) containing two lysis genes, did not confer a rapid decline in the culture turbidity following induction with mitomycin C. Interestingly a subset of the cells suffered perturbation of their outer membrane, which was not observed from single lysis mutant (∆celE or ∆celI) cells. This observed heterogeneity in the colicin E9 release leading to differential outer membrane perforation may bring a competitive advantage to these cells in a mixed population.

Genome-Wide Investigation of Oxidosqualene Cyclase Genes Deciphers the Genetic Basis of Triterpene Biosynthesis in Tea Plants.

Triterpenoids from Camellia species comprise a diverse class of bioactive compounds with great therapeutic potential. However, triterpene biosynthesis in tea plants (Camellia sinensis) remains elusive. Here, we identified eight putative 2,3-oxidosqualene cyclase (OSC) genes (CsOSC1-8) from the tea genome and characterized the functions of five through heterologous expression in yeast and tobacco and transient overexpression in tea plants. CsOSC1 was found to be a β-amyrin synthase, whereas CsOSC4, 5, and 6 exhibited multifunctional α-amyrin synthase activity. Molecular docking and site-directed mutagenesis showed that the CsOSC6M259T/W260L double mutant yielded >40% lupeol, while the CsOSC1 W259L single mutant alone was sufficient for lupeol production. The V732F mutation in CsOSC5 altered product formation from friedelin to taraxasterol and ψ-taraxasterol. The L254 M mutation in the cycloartenol synthase CsOSC8 enhanced the catalytic activity. Our findings shed light on the molecular basis governing triterpene diversity in tea plants and offer potential avenues for OSC engineering.

The significance of the two cytosolic glutamine synthetase enzymes, GLN1;3 and GLN1;5, in the context of seed development and germination in Arabidopsis thaliana

Glutamine synthetase (GS), an initial enzyme in nitrogen (N) plant metabolism, exists as a group of isoenzymes found in both cytosolic (GS1) and plastids (GS2) and has gathered significant attention for enhancing N use efficiency and crop yield.This work focuses on the A. thaliana GLN1;3 and GLN1;5 genes, the two predicted most expressed genes in seeds, among the five isogenes encoding GS1 in this species. The expression patterns were studied using transgenic marker line plants and qPCR during seed development and germination. The observed patterns highlight distinct functions for the two genes and confirm GLN1;5 as the most highly expressed GS1 gene in seeds. The GLN1;5, expression, oriented towards hypocotyl and cotyledons, suggests a role in protein turnover during germination, while the radicle-oriented expression of GLN1;3 supports a function in early external N uptake.While the single mutants exhibited a normal phenotype, except for a decrease in seed parameters, the double gln1;3/gln1;5 mutant displayed a germination delay, substantial impairment in growth, nitrogen metabolism, and number and quality of the seeds, as well as a diminishing in flowering. Although seed and pollen-specific, GLN1;5 expression is upregulated in the meristems of the gln1;3 mutants, filling the lack of GLN1;3 and ensuring the normal functioning of the gln1;3 mutants.These findings validate earlier in silico data on the expression patterns of GLN1;3 and GL1;5 genes in seeds, explore their different functions, and underscore their essential role in plant growth, seed production, germination, and early stages of plant development.

CRISPR/Cas9-mediated editing of GmDWF1 brassinosteroid biosynthetic gene induces dwarfism in soybean.

The study on the GmDWF1-deficient mutant dwf1 showed that GmDWF1 plays a crucial role in determining soybean plant height and yield by influencing the biosynthesis of brassinosteroids. Soybean has not adopted the Green Revolution, such as reduced height for increased planting density, which have proven beneficial for cereal crops. Our research identified the soybean genes GmDWF1a and GmDWF1b, homologous to Arabidopsis AtDWF1, and found that they are widely expressed, especially in leaves, and linked to the cellular transport system, predominantly within the endoplasmic reticulum and intracellular vesicles. These genes are essential for the synthesis of brassinosteroids (BR). Single mutants of GmDWF1a and GmDWF1b, as well as double mutants of both genes generated through CRISPR/Cas9 genome editing, exhibit a dwarf phenotype. The single-gene mutant exhibits moderate dwarfism, while the double mutant shows more pronounced dwarfism. Despite the reduced stature, all types of mutants preserve their node count. Notably, field tests have shown that the single GmDWF1a mutant produced significantly more pods than wild-type plants. Spraying exogenous brassinolide (BL) can compensate for the loss in plant height induced by the decrease in endogenous BRs. Comparing transcriptome analyses of the GmDWF1a mutant and wild-type plants revealed a significant impact on the expression of many genes that influence soybean growth. Identifying the GmDWF1a and GmDWF1b genes could aid in the development of compact, densely planted soybean varieties, potentially boosting productivity.

Hypoxia reshapes Arabidopsis root architecture by integrating ERF-VII factor response and abscisic acid homoeostasis.

Oxygen limitation (hypoxia), arising as a key stress factor due to flooding, negatively affects plant development. Consequently, maintaining root growth under such stress is crucial for plant survival, yet we know little about the root system's adaptions to low-oxygen conditions and its regulation by phytohormones. In this study, we examine the impact of hypoxia and, herein, the regulatory role of group VII ETHYLENE-RESPONSE FACTOR (ERFVII) transcription factors on root growth in Arabidopsis. We found lateral root (LR) elongation to be actively maintained by hypoxia via ERFVII factors, as erfVII seedlings possess hypersensitivity towards hypoxia regarding their LR growth. Pharmacological inhibition of abscisic acid (ABA) biosynthesis revealed ERFVII-driven counteraction of hypoxia-induced inhibition of LR formation in an ABA-dependent manner. However, postemergence LR growth under hypoxia mediated by ERFVIIs was independent of ABA. In roots, ERFVIIs mediate, among others, the induction of ABA-degrading ABA 8'-hydroxylases CYP707A1 expression. RAP2.12 could activate the pCYC707A1:LUC reporter gene, indicating, combined with single mutant analyses, that this transcription factor regulates ABA levels through corresponding transcript upregulation. Collectively, hypoxia-induced adaptation of the Arabidopsis root system is shaped by developmental reprogramming, whereby ERFVII-dependent promotion of LR emergence, but not elongation, is partly executed through regulation of ABA degradation.

BIG enhances Arg/N-degron pathway-mediated protein degradation to regulate Arabidopsis hypoxia responses and suberin deposition.

BIG/DARK OVEREXPRESSION OF CAB1/TRANSPORT INHIBITOR RESPONSE3 is a 0.5-MDa protein associated with multiple functions in Arabidopsis (Arabidopsis thaliana) signalling and development. However, the biochemical functions of BIG are unknown. We investigated a role for BIG in the Arg/N-degron pathways, in which substrate protein fate is influenced by the N-terminal (Nt) residue. We crossed a big loss-of-function allele to two N-degron pathway E3 ligase mutants, proteolysis6 (prt6) and prt1, and examined the stability of protein substrates. Stability of model substrates was enhanced in prt6-1 big-2 and prt1-1 big-2 relative to the respective single mutants and the abundance of the PRT6 physiological substrates, HYPOXIA-RESPONSIVE ERF2 (HRE2) and VERNALIZATION2 (VRN2) was similarly increased in prt6 big double mutants. Hypoxia marker expression was enhanced in prt6 big double mutants; this constitutive response required arginyltransferase activity and RAP-type ERFVII transcription factors. Transcriptomic analysis of roots not only demonstrated increased expression of multiple hypoxia-responsive genes in the double mutant relative to prt6, but also revealed other roles for PRT6 and BIG, including regulation of suberin deposition through both ERFVII-dependent and independent mechanisms, respectively. Our results show that BIG acts together with PRT6 to regulate the hypoxia response and broader processes in Arabidopsis.