Genetic engineering of drought- and salt-tolerant tomato via Δ1-pyrroline-5-carboxylate reductase S-nitrosylation.

Drought and soil salinization substantially impact agriculture. While proline's role in enhancing stress tolerance is known, the exact molecular mechanism by which plants process stress signals and control proline synthesis under stress is still not fully understood. In tomato (Solanum lycopersicum L.), drought and salt stress stimulate nitric oxide (NO) production, which boosts proline synthesis by activating Δ1-pyrroline-5-carboxylate synthetase (SlP5CS) and Δ1-pyrroline-5-carboxylate reductase (SlP5CR) genes and the P5CR enzyme. The crucial factor is stress-triggered NO production, which regulates the S-nitrosylation of SlP5CR at Cys-5, thereby increasing its NAD(P)H affinity and enzymatic activity. S-nitrosylation of SlP5CR enables tomato plants to better adapt to changing NAD(P)H levels, boosting both SlP5CR activity and proline synthesis during stress. By comparing tomato lines genetically modified to express different forms of SlP5CR, including a variant mimicking S-nitrosylation (SlP5CRC5W), we found that SlP5CRC5W plants show superior growth and stress tolerance. This is attributed to better P5CR activity, proline production, water use efficiency, reactive oxygen species scavenging, and sodium excretion. Overall, this study demonstrates that tomato engineered to mimic S-nitrosylated SlP5CR exhibits enhanced growth and yield under drought and salt stress conditions, highlighting a promising approach for stress-tolerant tomato cultivation.

The UBP14-CDKB1;1-CDKG2 cascade controls endoreduplication and cell growth in Arabidopsis.

Endoreduplication, a process in which DNA replication occurs in the absence of mitosis, is found in all eukaryotic kingdoms, especially plants, where it is assumed to be important for cell growth and cell fate maintenance. However, a comprehensive understanding of the mechanism regulating endoreduplication is still lacking. We previously reported that UBIQUITIN-SPECIFIC PROTEASE14 (UBP14), encoded by DA3, acts upstream of CYCLIN-DEPENDENT KINASE B1;1 (CDKB1;1) to influence endoreduplication and cell growth in Arabidopsis thaliana. The da3-1 mutant possesses large cotyledons with enlarged cells due to high ploidy levels. Here, we identified a suppressor of da3-1 (SUPPRESSOR OF da3-1 6; SUD6), encoding CYCLIN-DEPENDENT KINASE G2 (CDKG2), which promotes endoreduplication and cell growth. CDKG2/SUD6 physically associates with CDKB1;1 in vivo and in vitro. CDKB1;1 directly phosphorylates SUD6 and modulates its stability. Genetic analysis indicated that SUD6 acts downstream of DA3 and CDKB1;1 to control ploidy level and cell growth. Thus, our study establishes a regulatory cascade for UBP14/DA3-CDKB1;1-CDKG2/SUD6-mediated control of endoreduplication and cell growth in Arabidopsis.

Redesigning a S-nitrosylated pyruvate-dependent GABA transaminase 1 to generate high-malate and saline-alkali-tolerant tomato.

Although saline-alkali stress can improve tomato quality, the detailed molecular processes that balance stress tolerance and quality are not well-understood. Our research links nitric oxide (NO) and γ-aminobutyric acid (GABA) with the control of root malate exudation and fruit malate storage, mediated by aluminium-activated malate transporter 9/14 (SlALMT9/14). By modifying a specific S-nitrosylated site on pyruvate-dependent GABA transaminase 1 (SlGABA-TP1), we have found a way to enhance both plant's saline-alkali tolerance and fruit quality. Under saline-alkali stress, NO levels vary in tomato roots and fruits. High NO in roots leads to S-nitrosylation of SlGABA-TP1/2/3 at Cys316/258/316, reducing their activity and increasing GABA. This GABA then reduces malate exudation from roots and affects saline-alkali tolerance by interacting with SlALMT14. In fruits, a moderate NO level boosts SlGABA-TP1 expression and GABA breakdown, easing GABA's block on SlALMT9 and increasing malate storage. Mutants of SlGABA-TP1C316S that do not undergo S-nitrosylation maintain high activity, supporting malate movement in both roots and fruits under stress. This study suggests targeting SlGABA-TP1Cys316 in tomato breeding could significantly improve plant's saline-alkali tolerance and fruit quality, offering a promising strategy for agricultural development.

High-nitrogen-induced γ-aminobutyric acid triggers host immunity and pathogen oxidative stress tolerance in tomato and Ralstonia solanacearum interaction.

Soil nitrogen (N) significantly influences the interaction between plants and pathogens, yet its impact on host defenses and pathogen strategies via alterations in plant metabolism remains unclear. Through metabolic and genetic studies, this research demonstrates that high-N-input exacerbates tomato bacterial wilt by altering γ-aminobutyric acid (GABA) metabolism of host plants. Under high-N conditions, the nitrate sensor NIN-like protein 7 (SlNLP7) promotes the glutamate decarboxylase 2/4 (SlGAD2/4) transcription and GABA synthesis by directly binding to the promoters of SlGAD2/4. The tomato plants with enhanced GABA levels showed stronger immune responses but remained susceptible to Ralstonia solanacearum. This led to the discovery that GABA produced by the host actually heightens the pathogen's virulence. We identified the R. solanacearum LysR-type transcriptional regulator OxyR protein, which senses host-derived GABA and, upon interaction, triggers a response involving protein dimerization that enhances the pathogen's oxidative stress tolerance by activating the expression of catalase (katE/katGa). These findings reveal GABA's dual role in activating host immunity and enhancing pathogen tolerance to oxidative stress, highlighting the complex relationship between tomato plants and R. solanacearum, influenced by soil N status.

S-nitrosylation of ACO homolog 4 improves ethylene synthesis and salt tolerance in tomato.

Crop loss due to soil salinization is a global threat to agriculture. Nitric oxide (NO) and ethylene involve in multiple plant tolerance. However, their interaction in salt resistance remains largely elusive. We tested the mutual induction between NO and ethylene, and then identified an 1-aminocyclopropane-1-carboxylate oxidase homolog 4 (ACOh4) that influences ethylene synthesis and salt tolerance through NO-mediated S-nitrosylation. Both NO and ethylene positively responded to salt stress. Furthermore, NO participated in salt-induced ethylene production. Salt tolerance evaluation showed that function of NO was abolished by inhibiting ethylene production. Conversely, function of ethylene was little influenced by blocking NO generation. ACO was identified as the target of NO to control ethylene synthesis. In vitro and in vivo results suggested that ACOh4 was S-nitrosylated at Cys172, resulting in its enzymatic activation. Moreover, ACOh4 was induced by NO through transcriptional manner. Knockdown of ACOh4 abolished NO-induced ethylene production and salt tolerance. At physiological status, ACOh4 positively regulates the Na+ and H+ efflux, and keeps K+ /Na+ homeostasis by promoting salt-resistive genes' transcripts. Our findings validate a role of NO-ethylene module in salt tolerance and uncover a novel mechanism of how NO promoting ethylene synthesis against adversity.

A Large-Scale Mutational Analysis of Two-Component Signaling Systems of Lonsdalea quercina Revealed that KdpD-KdpE Regulates Bacterial Virulence Against Host Poplar Trees.

Poplar, which is a dominant species in plant communities distributed in the northern hemisphere, is commonly used as a model plant in forestry studies. Poplar production can be inhibited by infections caused by bacteria, including Lonsdalea quercina subsp. populi, which is a gram-negative bacterium responsible for bark canker disease. However, the molecular basis of the pathogenesis remains uncharacterized. In this study, we annotated the two-component signal transduction systems (TCSs) encoded by the L. quercina subsp. populi N-5-1 genome and identified 18 putative histidine kinases and 24 response regulators. A large-scale mutational analysis revealed that 19 TCS genes regulated bacterial virulence against poplar trees. Additionally, the deletion of kdpE or overexpression of kdpD resulted in almost complete loss of bacterial virulence. We observed that kdpE and kdpD formed a bi-cistronic operon. KdpD exhibited autokinase activity and could bind to KdpE (Kd = 5.73 ± 0.64 µM). Furthermore, KdpE is an OmpR family response regulator. A chromatin immunoprecipitation sequencing analysis revealed that KdpE binds to an imperfect palindromic sequence within the promoters of 44 genes, including stress response genes Lqp0434, Lqp3037, and Lqp3270. A comprehensive analysis of TCS functions may help to characterize the regulation of poplar bark canker disease.

A PCR-free screen-printed magnetic electrode for the detection of circular RNA from hepatocellular cancer based on a back-splice junction.

Circular RNA (circRNA) has the potential to be applied to disease diagnosis and therapy. However, the currently available circRNA detection techniques are limited. This work proposes a sensitive and selective approach for circRNA detection based on gold nanoparticle-modified screen-printed magnetic electrodes (AuNPs-SPME). Magnetic beads (MBs) with capture probes based on specific back-splice junction (BSJ) sites were employed to identify and selectively isolate the target circRNA, which could be directly adsorbed onto the AuNPs-SPME. Then, the circRNA attached to the surface was detected by changes in the methylene blue redox signal. The simple and time-saving AuNPs-SPME is highly sensitive (LOD = 1.0 pM) to circCDYL, one of the biomarkers of hepatocellular cancer (HCC). The analytical performance of the method presented has also been verified in human serum samples, holding great promise for clinical diagnosis.

Facile one-pot solvothermal synthesis of magnetic mesoporous carbon for the efficient adsorption of methyl orange.

A facile one-pot solvent thermal method was proposed to synthesize magnetic mesoporous carbon (MMC) using Fe(NO3)3·9H2O as a precursor, Pluronic copolymer P123 as template, and chitosan as carbon source, and it was applied for the adsorptive remediation of methyl orange (MO). The characterization results of TEM, XRD, and IR showed that MMC consisted of graphitized carbon matrix and some black spherical particle mixture of Fe3O4 and Fe, and it was rich in hydroxyl and carbonyl groups. Besides, the effect of the content of Fe and the content of chitosan in MMC on the magnetism and adsorption performance of prepared material were investigated. In addition, the effects of pH value, initial concentration of methyl orange, and contact time on the adsorption performance of MO were studied, respectively. At 318 K, the maximum adsorption capacity of MO calculated from Langmuir isotherm was from 139 to 400 mg g-1 on MMC. Kinetic studies demonstrated that the adsorption process obeyed a pseudo-second-order kinetic model. The regeneration experiments revealed that MMC could be reused at least five times without notable decrease of adsorption performance. These results illustrate that MMC is an efficient and economical adsorbent for the adsorption of MO.

Analysis of HrpG regulons and HrpG-interacting proteins by ChIP-seq and affinity proteomics in Xanthomonas campestris.

Gamma-proteobacteria Xanthomonas spp. cause at least 350 different plant diseases among important agricultural crops, which result in serious yield losses. Xanthomonas spp. rely mainly on the type III secretion system (T3SS) to infect their hosts and induce a hypersensitive response in nonhosts. HrpG, the master regulator of the T3SS, plays the dominant role in bacterial virulence. In this study, we used chromatin immunoprecipitation followed by sequencing (ChIP-seq) and tandem affinity purification (TAP) to systematically characterize the HrpG regulon and HrpG interacting proteins in vivo. We obtained 186 candidate HrpG downstream genes from the ChIP-seq analysis, which represented the genomic-wide regulon spectrum. A consensus HrpG-binding motif was obtained and three T3SS genes, hpa2, hrcU, and hrpE, were confirmed to be directly transcriptionally activated by HrpG in the inducing medium. A total of 273 putative HrpG interacting proteins were identified from the TAP data and the DNA-binding histone-like HU protein of Xanthomonas campestris pv. campestris (HUxcc ) was proved to be involved in bacterial virulence by increasing the complexity and intelligence of the bacterial signalling pathways in the T3SS.

3D bimetallic Au/Pt nanoflowers decorated needle-type microelectrode for direct in situ monitoring of ATP secreted from living cells.

Adenosine triphosphate (ATP) plays a crucial role in energy metabolism and extracellular purinergic signaling. A 3D bimetallic Au/Pt nanoflowers decorated ATP microelectrode biosensor prepared by facile and effective template-free electrodeposition was firstly reported, realizing local detection of cellular ATP secretion. The ATP biosensor was developed by co-immobilization of glucose oxidase and hexokinase, exhibiting long-term stability (79.39 ± 9.15% of its initial value remained after 14 days at 4 °C) and high selectivity with a limit of detection down to 2.5 µM (S/N = 3). The resulting ATP biosensor was then used for direct in situ monitoring of ATP secreted from living cells (PC12) with the stimulation of high K+ solutions. The obtained current was about 21.6 ± 3.4 nA (N = 6), corresponding to 12.2 ± 2.8 µM ATP released from cells, right in the micromolar range and consistent with the suggested levels. The 3D bimetallic Au/Pt nanoflowers possess excellent catalytic activity and large electroactive surface area, contributing to enzymatic activity preservation and long-term stability. This work provides a promising platform for long-time monitoring of other neurotransmitters and secretions in cellular glycolysis and apoptosis processes in the future.

Three-dimensional paper-based microfluidic electrochemical integrated devices (3D-PMED) for wearable electrochemical glucose detection.

Wearable electrochemical sensors have attracted tremendous attention in recent years. Here, a three-dimensional paper-based microfluidic electrochemical integrated device (3D-PMED) was demonstrated for real-time monitoring of sweat metabolites. The 3D-PMED was fabricated by wax screen-printing patterns on cellulose paper and then folding the pre-patterned paper four times to form five stacked layers: the sweat collector, vertical channel, transverse channel, electrode layer and sweat evaporator. A sweat monitoring device was realized by integrating a screen-printed glucose sensor on polyethylene terephthalate (PET) substrate with the fabricated 3D-PMED. The sweat flow process in 3D-PMED was modelled with red ink to demonstrate the capability of collecting, analyzing and evaporating sweat, due to the capillary action of filter paper and hydrophobicity of wax. The glucose sensor was designed with a high sensitivity (35.7 µA mM-1 cm-2) and low detection limit (5 µM), considering the low concentration of glucose in sweat. An on-body experiment was carried out to validate the practicability of the three-dimensional sweat monitoring device. Such a 3D-PMED can be readily expanded for the simultaneous monitoring of alternative sweat electrolytes and metabolites.

High-Resolution Rapid Prototyping of Liquid Metal Electronics by Direct Writing on Highly Prestretched Substrates.

A rapid and inexpensive method to produce high-resolution liquid metal patterns and electronics on stretchable substrates was introduced. Two liquid-phase gallium-indium (GaIn) alloy patterns, conductive lines, and interdigitated electrodes, were directly written or shadow mask-printed on a prestretched elastomeric substrate surface. Then, the prestretched substrate was released to recover its original length, and thus, electronic patterns simultaneously shrank on it. After these patterns were transferred to another prestretched substrate by the stamp printing method, the patterning resolution was demonstrated to increase by totally 50 times for the two successive stretch-release-shrink operations. Additionally, the resistance of the handwritten liquid metal conductive line traces remained nearly unchanged during the stretching process, which is believed to be feasible for electrical connections in stretchable electronics. The rapid prototyping of a serpentine strain sensor was successfully demonstrated to be highly sensitive and repeatable with a stretching ratio ranging from 0 to 200%. The proposed method paves a new way to fabricate stretchable electronic devices with high patterning resolution.

Author Correction: BsmR degrades c-di-GMP to modulate biofilm formation of nosocomial pathogen Stenotrophomonas maltophilia.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

BsmR degrades c-di-GMP to modulate biofilm formation of nosocomial pathogen Stenotrophomonas maltophilia.

c-di-GMP is a cellular second messenger that regulates diverse bacterial processes, including swimming, biofilm formation and virulence. However, in Stenotrophomonas maltophilia, a nosocomial pathogen that frequently infects immunodeficient or immunoincompetent patients, the regulatory function of c-di-GMP remains unclear. Here we show that BsmR is a negative regulator of biofilm development that degrades c-di-GMP through its EAL domain. Increasing BsmR expression resulted in significant increase in bacterial swimming and decrease in cell aggregation. BsmR regulates the expression of at least 349 genes. Among them, 34 involved in flagellar assembly and a flagellar-assembly-related transcription factor (fsnR) are positively regulated. Although BsmR is a response regulator of the two-component signaling system, its role in biofilm formation depends on the expression level of its respective gene (bsmR), not on the protein's phosphorylation level. A transcription factor, BsmT, whose coding gene is located in the same tetra-cistronic operon as bsmR, was shown to directly bind to the promoter region of the operon and, through a positive regulatory loop, modulate bsmR transcription. Thus, our results revealed that the c-di-GMP signaling pathway controls biofilm formation and swimming in S. maltophilia, suggesting c-di-GMP signaling as a target in the development of novel antibacterial agents to resist this pathogen.

An Essential Regulatory System Originating from Polygenic Transcriptional Rewiring of PhoP-PhoQ of Xanthomonas campestris.

How essential, regulatory genes originate and evolve is intriguing because mutations of these genes not only lead to lethality in organisms, but also have pleiotropic effects since they control the expression of multiple downstream genes. Therefore, the evolution of essential, regulatory genes is not only determined by genetic variations of their own sequences, but also by the biological function of downstream genes and molecular mechanisms of regulation. To understand the origin of essential, regulatory genes, experimental dissection of the complete regulatory cascade is needed. Here, we provide genetic evidences to reveal that PhoP-PhoQ is an essential two-component signal transduction system in the gram-negative bacterium Xanthomonas campestris, but that its orthologs in other bacteria belonging to Proteobacteria are nonessential. Mutational, biochemical, and chromatin immunoprecipitation together with high-throughput sequencing analyses revealed that phoP and phoQ of X. campestris and its close relative Pseudomonas aeruginosa are replaceable, and that the consensus binding motifs of the transcription factor PhoP are also highly conserved. PhoP Xcc in X. campestris regulates the transcription of a number of essential, structural genes by directly binding to cis-regulatory elements (CREs); however, these CREs are lacking in the orthologous essential, structural genes in P. aeruginosa, and thus the regulatory relationships between PhoP Pae and these downstream essential genes are disassociated. Our findings suggested that the recruitment of regulatory proteins by critical structural genes via transcription factor-CRE rewiring is a driving force in the origin and functional divergence of essential, regulatory genes.