Arabidopsis Seedlings Research Articles

RHA2b-mediated MYB30 degradation facilitates MYB75-regulated, sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings

Anthocyanins play diverse roles in plant physiology and stress adaptation. In Arabidopsis, the MYB–bHLH–WD40 (MBW) complex has a crucial role in the regulation of anthocyanin synthesis. Here, we report that the R2R3-MYB transcription factor MYB30 and the ubiquitin E3 ligase RHA2b participate in anthocyanin biosynthesis through regulation of the MBW complex. MYB30 was found to negatively regulate sucrose-induced anthocyanin biosynthesis in Arabidopsis seedlings. Expression of multiple genes involved in flavonoid or anthocyanin biosynthesis was affected in the myb30 mutant, and MYB30 directly repressed the expression of MYB75, which encodes a core component of the MBW complex, by binding to its promoter. Moreover, MYB30 physically interacted with MYB75 to inhibit its activity by repressing MBW complex assembly. In addition, sucrose treatment significantly promoted MYB30 degradation via the action of RHA2b. The ubiquitination and degradation of MYB30 were significantly attenuated in the rha2b mutant under high-sucrose treatment, and further analysis showed that MYB75 directly promoted RHA2b expression in response to high sucrose. Our work thus reveals an anthocyanin biosynthetic regulatory module, RHA2b–MYB30, that controls the function of the MBW complex via MYB75. The repression of MYB75 by MYB30 is released by MYB75-induced RHA2b expression, thus ensuring the self-activation of MYB75 when anthocyanin synthesis is needed.

Phosphoproteome analyses pinpoint the F-box protein SLOW MOTION as a regulator of warm temperature-mediated hypocotyl growth in Arabidopsis.

Hypocotyl elongation is controlled by several signals and is a major characteristic of plants growing in darkness or under warm temperature. While already several molecular mechanisms associated with this process are known, protein degradation and associated E3 ligases have hardly been studied in the context of warm temperature. In a time-course phosphoproteome analysis on Arabidopsis seedlings exposed to control or warm ambient temperature, we observed reduced levels of diverse proteins over time, which could be due to transcription, translation, and/or degradation. In addition, we observed differential phosphorylation of the LRR F-box protein SLOMO MOTION (SLOMO) at two serine residues. We demonstrate that SLOMO is a negative regulator of hypocotyl growth, also under warm temperature conditions, and protein-protein interaction studies revealed possible interactors of SLOMO, such as MKK5, DWF1, and NCED4. We identified DWF1 as a likely SLOMO substrate and a regulator of warm temperature-mediated hypocotyl growth. We propose that warm temperature-mediated regulation of SLOMO activity controls the abundance of hypocotyl growth regulators, such as DWF1, through ubiquitin-mediated degradation.

Comprehensive analysis of the UDP-glucuronate decarboxylase (UXS) gene family in tobacco and functional characterization of NtUXS16 in Golgi apparatus in Arabidopsis

BackgroundUDP-glucuronate decarboxylase (also named UXS) converts UDP-glucuronic acid (UDP-GlcA) to UDP-xylose (UDP-Xyl) by decarboxylation of the C6-carboxylic acid of glucuronic acid. UDP-Xyl is an important sugar donor that is required for the synthesis of plant cell wall polysaccharides.ResultsIn this study, we first carried out the genome-wide identification of NtUXS genes in tobacco. A total of 17 NtUXS genes were identified, which could be divided into two groups (Group I and II), and the Group II UXSs can be further divided into two subgroups (Group IIa and IIb). Furthermore, the protein structures, intrachromosomal distributions and gene structures were thoroughly analyzed. To experimentally verify the subcellular localization of NtUXS16 protein, we transformed tobacco BY-2 cells with NtUXS16 fused to the monomeric red fluorescence protein (mRFP) at the C terminus under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The fluorescent signals of NtUXS16-mRFP were localized to the medial-Golgi apparatus. Contrary to previous predictions, protease digestion analysis revealed that NtUXS16 is not a type II membrane protein. Overexpression of NtUXS16 in Arabidopsis seedling in darkness led to a significant increase in hypocotyl length and a reduction in root length compared with the wild type. In summary, these results suggest Golgi apparatus localized-NtUXS16 plays an important role in hypocotyl and root growth in the dark.ConclusionOur findings facilitate our understanding of the novel functions of NtUXS16 and provide insights for further exploration of the biological roles of NtUXS genes in tobacco.

Inter-Organellar Effects ofDefective ER-Localized Linolenic Acid Formation onThylakoid Lipid Composition, Non-Photochemical Quenching ofChlorophyll Fluorescence andXanthophyll Cycle Activity inthe Arabidopsis fad3 Mutant.

Monogalactosyldiacylglycerol (MGDG) is the main lipid constituent of thylakoids and a structural component of photosystems and photosynthesis-related proteo-lipid complexes in green tissues. Previously reported changes in MGDG abundance upon stress treatments are hypothesized to reflect mobilization of MGDG-based polyunsaturated lipid intermediates to maintain extraplastidial membrane integrity. While exchange of lipid intermediates between compartmental membranes is well documented, physiological consequences of mobilizing an essential thylakoid lipid, such as MGDG, for an alternative purpose are not well understood. Arabidopsis seedlings exposed to mild (50 mM) salt treatment displayed significantly increased abundance of both MGDG and the extraplastidial lipid, phosphatidylcholine (PC). Interestingly, similar increases in MGDG and PC were observed in Arabidopsis fad3 mutant seedlings defective in endoplasmic reticulum (ER)-localized linolenic acid formation, in which compensatory plastid-to-ER-directed mobilization of linolenic acid-containing intermediates takes place. The postulated (salt) or evident (fad3) plastid-ER exchange of intermediates concurred with altered thylakoid function according to parameters of photosynthetic performance. While salt treatment of wild-type seedlings inhibited photosynthetic parameters in a dose-dependent manner, interestingly, untreated fad3 mutants did not show overall reduced photosynthetic quantum yield. By contrast, we observed a reduction specifically of non-photochemical quenching (NPQ) under high light, representing only part of observed salt effects. The decreased NPQ in the fad3 mutant was accompanied by reduced activity of the xanthophyll cycle, leading to a reduced concentration of the NPQ-effective pigment zeaxanthin. The findings suggest that altered ER-located fatty acid unsaturation and ensuing inter-organellar compensation impacts on the function of specific thylakoid enzymes, rather than globally affecting thylakoid function.

Modulation of warm temperature-sensitive growth using a phytochrome B dark reversion variant, phyB[G515E], in Arabidopsis and rice

IntroductionAmbient temperature-induced hypocotyl elongation in Arabidopsis seedlings is sensed by the epidermis-localized phytochrome B (phyB) and transduced into auxin biosynthesis via a basic helix-loop-helix transcription factor, phytochrome-interacting factor 4 (PIF4). Once synthesized, auxin travels down from the cotyledons to the hypocotyl, triggering hypocotyl cell elongation. Thus, the phyB–PIF4 module involved in thermosensing and signal transduction is a potential genetic target for engineering warm temperature-insensitive plants. ObjectivesThis study aims to manipulate warm temperature-induced elongation of plants at the post-translational level using phyB variants with dark reversion, the expression of which is subjected to heat stress. MethodsThe thermosensitive growth response of Arabidopsis was manipulated by expressing the single amino acid substitution variant of phyB (phyB[G515E]), which exhibited a lower dark reversion rate than wild-type phyB. Other variants with slow (phyB[G564E]) or rapid (phyB[S584F]) dark reversion or light insensitivity (phyB[G767R]) were also included in this study for comparison. Warming-induced transient expression of phyB variants was achieved using heat shock-inducible promoters. Arabidopsis PHYB[G515E] and PHYB[G564E] were also constitutively expressed in rice in an attempt to manipulate the heat sensitivity of a monocotyledonous plant species. ResultsAt an elevated temperature, Arabidopsis seedlings transiently expressing PHYB[G515E] under the control of a heat shock-inducible promoter exhibited shorter hypocotyls than those expressing PHYB and other PHYB variant genes. This warm temperature-insensitive growth was related to the lowered PIF4 and auxin responses. In addition, transgenic rice seedlings expressing Arabidopsis PHYB[G515E] and PHYB[G564E] showed warm temperature-insensitive shoot growth. ConclusionTransient expression of phyB variants with altered dark reversion rates could serve as an effective optogenetic technique for manipulating PIF4–auxin-mediated thermomorphogenic responses in plants.

Transcriptional reprogramming of nucleotide metabolism in response to altered pyrimidine availability in Arabidopsis seedlings.

In Arabidopsis seedlings, inhibition of aspartate transcarbamoylase (ATC) and de novo pyrimidine synthesis resulted in pyrimidine starvation and developmental arrest a few days after germination. Synthesis of pyrimidine nucleotides by salvaging of exogenous uridine (Urd) restored normal seedling growth and development. We used this experimental system and transcriptional profiling to investigate genome-wide responses to changes in pyrimidine availability. Gene expression changes at different times after Urd supplementation of pyrimidine-starved seedlings were mapped to major pathways of nucleotide metabolism, in order to better understand potential coordination of pathway activities, at the level of transcription. Repression of de novo synthesis genes and induction of intracellular and extracellular salvaging genes were early and sustained responses to pyrimidine limitation. Since de novo synthesis is energetically more costly than salvaging, this may reflect a reduced energy status of the seedlings, as has been shown in recent studies for seedlings growing under pyrimidine limitation. The unexpected induction of pyrimidine catabolism genes under pyrimidine starvation may result from induction of nucleoside hydrolase NSH1 and repression of genes in the plastid salvaging pathway, diverting uracil (Ura) to catabolism. Identification of pyrimidine-responsive transcription factors with enriched binding sites in highly coexpressed genes of nucleotide metabolism and modeling of potential transcription regulatory networks provided new insights into possible transcriptional control of key enzymes and transporters that regulate nucleotide homeostasis in plants.

Finding a good balance: two distinct chromatin factors fine-tune stress response in Arabidopsis seedlings.

This article is a Commentary on Perrella et al. (2023), 241: 166–179.

Time series single-cell transcriptional atlases reveal cell fate differentiation driven by light in Arabidopsis seedlings

Light serves as the energy source for plants as well as a signal for growth and development during their whole life cycle. Seedling de-etiolation is the most dramatic manifestation of light-regulated plant development processes, as massive reprogramming of the plant transcriptome occurs at this time. Although several studies have reported about organ-specific development and expression induced by light, a systematic analysis of cell-type-specific differentiation and the associated transcriptional regulation is still lacking. Here we obtained single-cell transcriptional atlases for etiolated, de-etiolating and light-grown Arabidopsisthaliana seedlings. Informative cells from shoot and root tissues were grouped into 48 different cell clusters and finely annotated using multiple markers. With the determination of comprehensive developmental trajectories, we demonstrate light modulation of cell fate determination during guard cell specialization and vasculature development. Comparison of expression atlases between wild type and the pifq mutant indicates that phytochrome-interacting factors (PIFs) are involved in distinct developmental processes in endodermal and stomatal lineage cells via controlling cell-type-specific expression of target genes. These results provide information concerning the light signalling networks at the cell-type resolution, improving our understanding of how light regulates plant development at the cell-type and genome-wide levels. The obtained information could serve as a valuable resource for comprehensively investigating the molecular mechanism of cell development and differentiation in response to light.

Arabidopsis reticulons inhibit ROOT HAIR DEFECTIVE3 to form a stable tubular endoplasmic reticulum network.

The endoplasmic reticulum (ER) is a network of interconnected tubules and sheets stretching throughout the cytoplasm of plant cells. In Arabidopsis (Arabidopsis thaliana), ROOT HAIR DEFECTIVE3 (RHD3) mediates ER tubule fusion, while reticulon proteins induce ER membrane curvature to produce ER tubules. However, it is unclear if and how RHD3-reticulon interplay during the formation of the interconnected tubular ER network. We discovered that RHD3 physically interacts with Arabidopsis reticulon proteins, including reticulon-like protein subfamily B3 (RTNLB3), on ER tubules and at 3-way junctions of the ER. The RTNLB3 protein is widely expressed in Arabidopsis seedlings and localizes to ER tubules. Although the growth of knockout rtnlb3 mutant plants was relatively normal, root hairs of rtnlb3 were shorter than those of wild type. The ER in mature mutant cells was also more sheeted than that in wild type. rhd3 is known to have short roots and root hairs and less branched ER tubules in cells. Interestingly, rtnlb3 genetically antagonizes rhd3 in plant root development and in ER interconnectivity. We show that reticulons including RTNLB3 inhibit the ER fusion activity of RHD3, partly by interfering with RHD3 dimerization. We conclude that reticulon proteins negatively regulate RHD3 to balance its ER fusion activity for the formation of a stable tubular ER network in plant cell growth.

Identification of novel plant cysteine oxidase inhibitors from a yeast chemical genetic screen

Hypoxic responses in plants involve Plant Cysteine Oxidases (PCOs). They catalyze the N-terminal cysteine oxidation of Ethylene Response Factors VII (ERF-VII) in an oxygen-dependent manner, leading to their degradation via the cysteine N-degron pathway (Cys-NDP) in normoxia. In hypoxia, PCO activity drops, leading to the stabilization of ERF-VIIs and subsequent hypoxic gene upregulation. Thus far, no chemicals have been described to specifically inhibit PCO enzymes. In this work, we devised an invivo pipeline to discover Cys-NDP effector molecules. Budding yeast expressing AtPCO4 and plant-based ERF-VII reporters was deployed to screen a library of natural-like chemical scaffolds and was further combined with an Arabidopsis Cys-NDP reporter line. This strategy allowed us to identify three PCO inhibitors, two of which were shown to affect PCO activity invitro. Application of these molecules to Arabidopsis seedlings led to an increase in ERF-VII stability, induction of anaerobic gene expression, and improvement of tolerance to anoxia. By combining a high-throughput heterologous platform and the plant model Arabidopsis, our synthetic pipeline provides a versatile system to study how the Cys-NDP is modulated. Its first application here led to the discovery of at least two hypoxia-mimicking molecules with the potential to impact plant tolerance to low oxygen stress.

Volatile Compounds Emitted by Plant Growth-Promoting Fungus Tolypocladium inflatum GT22 Alleviate Copper andPathogen Stress.

Previous studies on the intricate interactions between plants and microorganisms have revealed that fungal volatile compounds (VCs) can affect plant growth and development. However, the precise mechanisms underlying these actions remain to be delineated. In this study, we discovered that VCs from the soilborne fungus Tolypocladium inflatum GT22 enhance the growth of Arabidopsis. Remarkably, priming Arabidopsis with GT22 VCs caused the plant to display an enhanced immune response and mitigated the detrimental effects of both pathogenic infections and copper stress. Transcriptomic analyses of Arabidopsis seedlings treated with GT22 VCs for 3, 24 and 48 h revealed that 90, 83 and 137 genes were differentially expressed, respectively. The responsive genes are known to be involved in growth, hormone regulation, defense mechanisms and signaling pathways. Furthermore, we observed the induction of genes related to innate immunity, hypoxia, salicylic acid biosynthesis and camalexin biosynthesis by GT22 VCs. Among the VCs emitted by GT22, exposure of Arabidopsis seedlings to limonene promoted plant growth and attenuated copper stress. Thus, limonene appears to be a key mediator of the interaction between GT22 and plants. Overall, our findings provide evidence that fungal VCs can promote plant growth and enhance both biotic and abiotic tolerance. As such, our study suggests that exposure of seedlings to T. inflatum GT22 VCs may be a means of improving crop productivity. This study describes a beneficial interaction between T. inflatun GT22 and Arabidopsis. Our investigation of microorganism function in terms of VC activities allowed us to overcome the limitations of traditional microbial application methods. The importance of this study lies in the discovery of T. inflatun GT22 as a beneficial microorganism. This soilborne fungus emits VCs with plant growth-promoting effects and the ability to alleviate both copper and pathogenic stress. Furthermore, our study offers a valuable approach to tracking the activities of fungal VC components via transcriptomic analysis and sheds light on the mechanisms through which VCs promote plant growth and induce resistance. This research significantly advances our knowledge of VC applications and provides an example for further investigations within this field.

The promoters of two CpMYB106-like paralog genes respond to abiotic stresses and phytohormones and drive differential expression in contrasting cultivars of Cucurbita pepo

Zucchini fruit (Cucurbita pepo L.) is susceptible to chilling injury (CI) during its postharvest life, with this response being cultivar-dependent. A previous transcriptomic analysis comparing cold-tolerant fruit from the cultivar ‘Natura’ with cold-sensitive fruit from the cultivar ‘Sinatra’, revealed the transcription factor (TF) CpMYB106-like as a relevant candidate gene for the acquisition of postharvest low-temperature tolerance. C. pepo has two CpMYB106-like paralogs, which are differentially present in the ‘Natura’ (CpMYB106-likeA) and ‘Sinatra’ (CpMYB106-likeB) cultivars due to gene and promoter deletions. The aim of this study was to investigate the transcriptional regulation of the CpMYB106-like paralogs to unravel their role in the postharvest life of zucchini fruit. For this, gene expression was analyzed and promoter sequences and their activities were studied through reporter β-glucuronidase (GUS) gene analysis in heterologous systems. An expression analysis showed that CpMYB106-like mRNA levels were induced only in the cold-stored fruit of ‘Natura’. The analysis of the promoter sequences showed numerous cis-regulatory elements (CREs), many of which have a differential distribution or frequency between the two paralogs. Promoter basal activity was determined by transient transformation of Nicotiana benthamiana leaves, which showed a higher expression driven by the promoter from the cold-tolerant cultivar ‘Natura’, which could be explained by the presence of three additional copies of the enhancer element EECCRCAH1 in proMYB106A. The tissue-specific expression pattern, and the response to abiotic stresses and phytohormones controlled by CpMYB106-like promoters was analyzed in transgenic Arabidopsis thaliana plants. GUS activity was mainly detected in the vascular system, leaves, and inflorescences, especially in siliques. With respect to abiotic stresses, low temperature decreased GUS expression in Arabidopsis, but increased it in plants carrying the ‘Natura’ promoter after a 24-h acclimation period at 22 °C. Salt treatment induced GUS activity driven by both promoters in Arabidopsis seedlings, but it was higher with the ‘Natura’ promoter. Furthermore, methyl jasmonate (MeJA) also induced a sharp increase of GUS controlled by proMYB106A. These results suggest that the regulation of CpMYB106-like TFs in zucchini is promoter-driven, and reveal the involvement of these genes in the acquisition of cold tolerance in fruit during postharvest stress conditions.

Identification ofChemicals That Abrogate Folate-Dependent Inhibition ofStarch Accumulation inNon-Photosynthetic Plastids ofArabidopsis.

Folate, also known as vitamin B9, is an essential cofactor for a variety of enzymes and plays a crucial role in many biological processes. We previously reported that plastidial folate prevents starch biosynthesis triggered by the influx of sugar into non-starch-accumulating plastids, such as etioplasts, and chloroplasts under darkness; hence the loss of plastidial folate induces the accumulation of starch in plastids. To understand the molecular mechanism underlying this phenomenon, we screened our in-house chemical library and searched their derivatives to identify chemicals capable of inducing starch accumulation in etioplasts. The results revealed four chemicals, compounds #120 and #375 and their derivatives, compounds #120d and #375d, respectively. The derivative compounds induced starch accumulation in etioplasts and suppressed hypocotyl elongation in dark-grown Arabidopsis seedlings. They also inhibited the post-germinative growth of seedlings under illumination. All four chemicals contained the sulfonamide group as a consensus structure. The sulfonamide group is also found in sulfa drugs, which exhibit antifolate activity, and in sulfonylurea herbicides. Further analyses revealed that compound #375d induces starch accumulation by inhibiting folate biosynthesis. By contrast, compound #120d neither inhibited folate biosynthesis nor exhibited the herbicide activity. Protein and metabolite analyses suggest that compound #120d abrogates folate-dependent inhibition of starch accumulation in etioplasts by enhancing starch biosynthesis.

Chemical induction of hypocotyl rooting reveals extensive conservation of auxin signalling controlling lateral and adventitious root formation.

Upon exposure to light, etiolated Arabidopsis seedlings form adventitious roots (AR) along the hypocotyl. While processes underlying lateral root formation are studied intensively, comparatively little is known about the molecular processes involved in the initiation of hypocotyl AR. AR and LR formation were studied using a small molecule named Hypocotyl Specific Adventitious Root INducer (HYSPARIN) that strongly induces AR but not LR formation. HYSPARIN does not trigger rapid DR5-reporter activation, DII-Venus degradation or Ca2+ signalling. Transcriptome analysis, auxin signalling reporter lines and mutants show that HYSPARIN AR induction involves nuclear TIR1/AFB and plasma membrane TMK auxin signalling, as well as multiple downstream LR development genes (SHY2/IAA3, PUCHI, MAKR4 and GATA23). Comparison of the AR and LR induction transcriptome identified SAURs, AGC kinases and OFP transcription factors as specifically upregulated by HYSPARIN. Members of the SAUR19 subfamily, OFP4 and AGC2 suppress HYS-induced AR formation. While SAUR19 and OFP subfamily members also mildly modulate LR formation, AGC2 regulates only AR induction. Analysis of HYSPARIN-induced AR formation uncovers an evolutionary conservation of auxin signalling controlling LR and AR induction in Arabidopsis seedlings and identifies SAUR19, OFP4 and AGC2 kinase as novel regulators of AR formation.

Enhancement of sulfur metabolism and antioxidant machinery confers Bacillus sp. Jrh14-10–induced alkaline stress tolerance in plant

Alkaline stress is a major environmental challenge that restricts plant growth and agricultural productivity worldwide. Plant growth-promoting rhizobacteria (PGPR) can be used to effectively enhance plant abiotic stress in an environment-friendly manner. However, PGPR that can enhance alkalinity tolerance are not well-studied and the mechanisms by which they exert beneficial effects remain elusive. In this study, we isolated Jrh14-10from the rhizosphere soil of halophyte Halerpestes cymbalaria (Pursh) Green and found that it can produce indole-3-acetic acid (IAA) and siderophore. By 16S rRNA gene sequencing, it was classified as Bacillus licheniformis. Inoculation Arabidopsis seedlings with Jrh14-10 significantly increased the total fresh weight (by 148.1%), primary root elongation (by 1121.7%), and lateral root number (by 108.8%) under alkaline stress. RNA-Seq analysis showed that 3389 genes were up-regulated by inoculation under alkaline stress and they were associated with sulfur metabolism, photosynthetic system, and oxidative stress response. Significantly, the levels of Cys and GSH were increased by 144.3% and 48.7%, respectively, in the inoculation group compared to the control under alkaline stress. Furthermore, Jrh14-10 markedly enhanced the activities of antioxidant enzymes, resulting in lower levels of O2•–, H2O2, and MDA as well as higher levels of Fv/Fm in alkaline-treated seedlings. In summary, Jrh14-10 can improve alkaline stress resistance in seedlings which was accompanied by an increase in sulfur metabolism-mediated GSH synthesis and antioxidant enzyme activities. These results provide a mechanistic understanding of the interactions between a beneficial bacterial strain and plants under alkaline stress.

Effect of the Static Magnetic Fields on the Circadian Rhythm in Arabidopsis thaliana

The influence of geomagnetic field on various biological processes is certainly an interesting research field, and more work is beingperformed across the globe. However, the impact of varying magnetic flux density on plant circadian rhythms has not yet beencompletely investigated. Different researchers have mostly focused their attention on the molecular workings of circadian rhythms invarious plant systems and the circadian architecture of transcriptomes under varying conditions. The present investigation evaluatedthe influence of variable geomagnetic field on the expression of genes that are under the control of circadian rhythm, and checkedwhether the expression pattern of these genes with respect to time (circadian nature) under different magnetic flux density changesor not in Arabidopsis seedlings. This study examined the impact of varying magnetic flux densities on the mRNA expression levels ofsix genes in Arabidopsis thaliana during the final 33 hours of their total 120 hour growth period. A. thaliana seedlings were subjectedto four distinct magnetic flux densities (0, 25, 50, and 90 μT), and the abundance of transcripts for chlorophyll a/b binding protein 4,the large subunit of RuBisCO, rubisco activase, chalcone synthase, porphobilinogen synthase, and phytoene dehydrogenase genes wasexamined. While the present study’s findings lend credence to the idea that the aforementioned genes are differentially expressed inresponse to changes in magnetic flux density, it also proved that the circadian nature of these genes was largely unaffected, with theirexpression pattern remaining largely unaltered regardless of the strength of the magnetic field.

Trichoderma virens Big Ras GTPase-1, a molecular switch for transforming a mutualistic fungus to plants in a deleterious microbe

Trichoderma spp. are ascomycete filamentous fungi widely distributed worldwide that establish mutualistic relationships with plants by antagonizing phytopathogens in the rhizosphere and colonizing the plant roots, hence, promoting plant growth and triggering the systemic resistance against phytopathogens. During the first stages of root colonization by Trichoderma, plants recognize the fungus as an invader by inducing the plant defense system, including the production of reactive oxygen species (ROS). Some members of the small Ras GTPases regulate NADPH oxidases and, therefore, ROS production. However, their role in mutualistic microorganisms that colonize plant roots is poorly understood. It has been demonstrated that Trichoderma virens strains lacking TBRG-1, a member of a new family of the Ras GTPases, impair their biocontrol capability and behave like a pathogen on tomato seeds and seedlings. Here, we found that TBRG-1 is essential in T. virens to maintain the mutualistic relationship with plants because a mutant-lacking tbrg-1 gen could not induce plant growth in Arabidopsis and tomatoes. Furthermore, treatment of Arabidopsis seedlings with Δtbrg-1 induced strongly PR-1a, the systemic acquired resistance (SAR) marker gene at early times of the interaction, which correlated with enhanced foliar damage by Botrytis cinerea, resembling the behavior of a biotrophic phytopathogen. Additionally, leaves of plants treated with either T. virens wild-type (wt) or Δtbrg-1 and challenged or not with Botrytis showed ROS production to a different extent, as well as differential expression of cell detoxification-related genes, CAT1, and APX1. Root colonization assays of sid-2 and jar1 mutant lines affected in SAR and induced systemic resistance (ISR) by Δtbrg-1 and the wt strain, suggest an important role of both pathways in the recognition of the fungus and that TBRG-1 plays a pivotal role in root colonization. Furthermore, we found that TBRG-1 is a negative regulator of NoxR expression, which may impact the mutualistic interaction.

CysQuant: Simultaneous quantification of cysteine oxidation and protein abundance using data dependent or independent acquisition mass spectrometry

Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function. Accurate quantification of cysteine oxidation is therefore crucial for decoding protein redox regulation. Here, we present CysQuant, a novel approach for simultaneous quantification of cysteine oxidation degrees and protein abundancies. CysQuant involves light/heavy iodoacetamide isotopologues for differential labeling of reduced and reversibly oxidized cysteines analyzed by data-dependent acquisition (DDA) or data-independent acquisition mass spectrometry (DIA-MS). Using plexDIA with in silico predicted spectral libraries, we quantified an average of 18% cysteine oxidation in Arabidopsis thaliana by DIA-MS, including a subset of highly oxidized cysteines forming disulfide bridges in AlphaFold2 predicted structures. Applying CysQuant to Arabidopsis seedlings exposed to excessive light, we successfully quantified the well-established increased reduction of Calvin-Benson cycle enzymes and discovered yet uncharacterized redox-sensitive disulfides in chloroplastic enzymes. Overall, CysQuant is a highly versatile tool for assessing the cysteine modification status that can be widely applied across various mass spectrometry platforms and organisms.

MeGLYI-13, a Glyoxalase I Gene in Cassava, Enhances the Tolerance of Yeast and Arabidopsis to Zinc and Copper Stresses.

Although zinc and copper are the two essential nutrients necessary for plant growth, their excessive accumulation in soil not only causes environmental pollution but also seriously threatens human health and inhibits plant growth. The breeding of plants with novel zinc or copper toxicity tolerance capacities represents one strategy to address this problem. Glyoxalase I (GLYI) family genes have previously been suggested to be involved in the resistance to a wide range of abiotic stresses, including those invoked by heavy metals. Here, a MeGLYI-13 gene cloned from a cassava SC8 cultivar was characterized with regard to its potential ability in resistance to zinc or copper stresses. Sequence alignment indicated that MeGLYI-13 exhibits sequence differences between genotypes. Transient expression analysis revealed the nuclear localization of MeGLYI-13. A nuclear localization signal (NLS) was found in its C-terminal region. There are 12 Zn2+ binding sites and 14 Cu2+ binding sites predicted by the MIB tool, of which six binding sites were shared by Zn2+ and Cu2+. The overexpression of MeGLYI-13 enhanced both the zinc and copper toxicity tolerances of transformed yeast cells and Arabidopsis seedlings. Taken together, our study shows the ability of the MeGLYI-13 gene to resist zinc and copper toxicity, which provides genetic resources for the future breeding of plants resistant to zinc and copper and potentially other heavy metals.

Functional Meta-Analysis of the Proteomic Responses of Arabidopsis Seedlings to the Spaceflight Environment Reveals Multi-Dimensional Sources of Variability across Spaceflight Experiments.

The human quest for sustainable habitation of extraterrestrial environments necessitates a robust understanding of life's adaptability to the unique conditions of spaceflight. This study provides a comprehensive proteomic dissection of the Arabidopsis plant's responses to the spaceflight environment through a meta-analysis of proteomics data from four separate spaceflight experiments conducted on the International Space Station (ISS) in different hardware configurations. Raw proteomics LC/MS spectra were analyzed for differential expression in MaxQuant and Perseus software. The analysis of dissimilarities among the datasets reveals the multidimensional nature of plant proteomic responses to spaceflight, impacted by variables such as spaceflight hardware, seedling age, lighting conditions, and proteomic quantification techniques. By contrasting datasets that varied in light exposure, we elucidated proteins involved in photomorphogenesis and skotomorphogenesis in plant spaceflight responses. Additionally, with data from an onboard 1 g control experiment, we isolated proteins that specifically respond to the microgravity environment and those that respond to other spaceflight conditions. This study identified proteins and associated metabolic pathways that are consistently impacted across the datasets. Notably, these shared proteins were associated with critical metabolic functions, including carbon metabolism, glycolysis, gluconeogenesis, and amino acid biosynthesis, underscoring their potential significance in Arabidopsis' spaceflight adaptation mechanisms and informing strategies for successful space farming.