H3K9 methylation regulates heterochromatin silencing through incoherent feedforward loops.

Histone H3 lysine-9 methylation (H3K9me) is a hallmark of the condensed and transcriptionally silent heterochromatin. It remains unclear how H3K9me controls transcription silencing and how cells delimit H3K9me domains to avoid silencing essential genes. Here, using Arabidopsis genetic systems that induce H3K9me2 in genes and transposons de novo, we show that H3K9me2 accumulation paradoxically also causes the deposition of the euchromatic mark H3K36me3 by a SET domain methyltransferase, ASHH3. ASHH3-induced H3K36me3 confers anti-silencing by preventing the demethylation of H3K4me1 by LDL2, which mediates transcriptional silencing downstream of H3K9me2. These results demonstrate that H3K9me2 not only facilitates but orchestrates silencing by actuating antagonistic silencing and anti-silencing pathways, providing insights into the molecular basis underlying proper partitioning of chromatin domains and the creation of metastable epigenetic variation.

Genome-wide characterization and functional analysis of the melon TGA gene family in disease resistance through ectopic overexpression in Arabidopsis thaliana.

TGA-binding (TGA) transcription factors, characterized by the basic region/leucine zipper motif (bZIP), have been recognized as pivotal regulators in plant growth, development, and stress responses through their binding to the as-1 element. In this study, the TGA gene families in melon, watermelon, cucumber, pumpkin, and zucchini were comprehensively characterized, encompassing analyses of gene/protein structures, phylogenetic relationships, gene duplication events, and cis-acting elements in gene promoters. Upon transient expression in Nicotiana benthamiana, the melon CmTGAs, with typical bZIP and DOG1 domains, were observed to localize within the nucleus. Biochemical investigation revealed specific interactions between CmTGA2/3/5/8/9 and CmNPR3 or CmNPR4. The CmTGA genes exhibited differential expression patterns in melon plants in response to different hormones like salicylic acid, methyl jasmonate, and ethylene, as well as a fungal pathogen, Stagonosporopsis cucurbitacearum that causes gummy stem blight in melon. The overexpression of CmTGA3, CmTGA8, and CmTGA9 in Arabidopsis plants resulted in the upregulation of AtPR1 and AtPR5 expression, thereby imparting enhanced resistance to Pseudomonas syringae pv. Tomato DC3000. In contrast, the overexpression of CmTGA7 or CmTGA9 resulted in a compromised resistance to Botrytis cinerea, coinciding with a concomitant reduction in the expression levels of AtPDF1.2 and AtMYC2 following infection with B. cinerea. These findings shed light on the important roles of specific CmTGA genes in plant immunity, suggesting that genetic manipulation of these genes could be a promising avenue for enhancing plant immune responses.

Artificially designed synthetic promoter for a high level of salt induction using a cis-engineering approach.

This work aimed to design a synthetic salt-inducible promoter using a cis-engineering approach. The designed promoter (PS) comprises a minimal promoter sequence for basal-level expression and upstream cis-regulatory elements (CREs) from promoters of salinity-stress-induced genes. The copy number, spacer lengths, and locations of CREs were manually determined based on their occurrence within native promoters. The initial activity profile of the synthesized PS promoter in transiently transformed N. tabacum leaves shows a seven-fold, five-fold, and four-fold increase in reporter GUS activity under salt, drought, and abscisic acid stress, respectively, at the 24-h interval, compared to the constitutive CaMV35S promoter. Analysis of gus expression in stable Arabidopsis transformants showed that the PS promoter induces over a two-fold increase in expression under drought or abscisic acid stress and a five-fold increase under salt stress at 24- and 48-h intervals, compared to the CaMV35S promoter. The promoter PS exhibits higher and more sustained activity under salt, drought, and abscisic acid stress compared to the constitutive CaMV35S.

Probing the Effect of Mutations on Light Harvesting in CP29 by Transient Absorption and First-Principles Simulations.

Natural light harvesting is exceptionally efficient thanks to the local energy funnel created within light-harvesting complexes (LHCs). To understand the design principles underlying energy transport in LHCs, ultrafast spectroscopy is often complemented by mutational studies that introduce perturbations into the excitonic structure of the natural complexes. However, such studies may fall short of identifying all excitation energy transfer (EET) pathways and their changes upon mutation. Here, we show that a synergistic combination of first-principles calculations and ultrafast spectroscopy can give unprecedented insight into the EET pathways occurring within LHCs. We measured the transient absorption spectra of the minor CP29 complex of plants and of two mutants, systematically mapping the kinetic components seen in experiments to the simulated exciton dynamics. With our combined strategy, we show that EET in CP29 is surprisingly robust to the changes in the exciton states induced by mutations, explaining the versatility of plant LHCs.

Co-pyrolysis of chicken manure with tree bark for reduced biochar toxicity and enhanced plant growth in Arabidopsis thaliana.

Co-pyrolysis of chicken manure with tree bark was investigated to mitigate salinity and potentially toxic element (PTE) concentrations of chicken manure-derived biochar. The effect of tree bark addition (0, 25, 50, 75 and 100 wt%) on the biochar composition, surface functional groups, PTEs and polycyclic aromatic hydrocarbons (PAH) concentration in the biochar was evaluated. Biochar-induced toxicity was assessed using an in-house plant growth assay with Arabidopsis thaliana. This study shows that PTE concentrations can be controlled through co-pyrolysis. More than 50 wt% of tree bark must be added to chicken manure to reduce the concentrations below the European Biochar Certificate-AGRO (EBC-AGRO) threshold. However, the amount of PAH does not show a trend with tree bark addition. Furthermore, co-pyrolysis biochar promotes plant growth at different application concentrations, whereas pure application of 100 wt% tree bark or chicken manure biochar results in decreased growth compared to the reference. In addition, increased plant stress was observed for 100 wt% chicken manure biochar. These data indicate that co-pyrolysis of chicken manure and tree bark produces EBC-AGRO-compliant biochar with the potential to stimulate plant growth. Further studies need to assess the effect of these biochars in long-term growth experiments.

Glutamate Receptor-like (GLR) Family in Brassica napus: Genome-Wide Identification and Functional Analysis in Resistance to Sclerotinia sclerotiorum.

Plant glutamate receptor-like channels (GLRs) are homologs of animal ionotropic glutamate receptors. GLRs are critical in various plant biological functions, yet their genomic features and functions in disease resistance remain largely unknown in many crop species. Here, we report the results on a thorough genome-wide study of the GLR family in oilseed rape (Brassica napus) and their role in resistance to the fungal pathogen Sclerotinia sclerotiorum. A total of 61 GLRs were identified in oilseed rape. They comprised three groups, as in Arabidopsis thaliana. Detailed computational analyses, including prediction of domain and motifs, cellular localization, cis-acting elements, PTM sites, and amino acid ligands and their binding pockets in BnGLR proteins, unveiled a set of group-specific characteristics of the BnGLR family, which included chromosomal distribution, motif composition, intron number and size, and methylation sites. Functional dissection employing virus-induced gene silencing of BnGLRs in oilseed rape and Arabidopsis mutants of BnGLR homologs demonstrated that BnGLR35/AtGLR2.5 positively, while BnGLR12/AtGLR1.2 and BnGLR53/AtGLR3.2 negatively, regulated plant resistance to S. sclerotiorum, indicating that GLR genes were differentially involved in this resistance. Our findings reveal the complex involvement of GLRs in B. napus resistance to S. sclerotiorum and provide clues for further functional characterization of BnGLRs.

Participation of miR165a in the Phytochrome Signal Transduction in Maize (Zea mays L.) Leaves under Changing Light Conditions.

The involvement of the microRNA miR165a in the light-dependent mechanisms of regulation of target genes in maize (Zea mays) has been studied. The light-induced change in the content of free miR165a was associated with its binding by the AGO10 protein and not with a change in the rate of its synthesis from the precursor. The use of knockout Arabidopsis plants for the phytochrome A and B genes demonstrated that the presence of an active form of phytochrome B causes an increase in the level of the RNA-induced silencing miR165a complex, which triggers the degradation of target mRNAs. The two fractions of vesicles from maize leaves, P40 and P100 that bind miR165a, were isolated by ultracentrifugation. The P40 fraction consisted of larger vesicles of the size >0.170 µm, while the P100 fraction vesicles were <0.147 µm. Based on the quantitative PCR data, the predominant location of miR165a on the surface of extracellular vesicles of both fractions was established. The formation of the active form of phytochrome upon the irradiation of maize plants with red light led to a redistribution of miR165a, resulting in an increase in its proportion inside P40 vesicles and a decrease in P100 vesicles.

Genome-Wide Analysis of MADS-Box Gene Family Reveals CjSTK as a Key Regulator of Seed Abortion in Camellia japonica.

The plant MADS-box transcription factor family is a major regulator of plant flower development and reproduction, and the AGAMOUS-LIKE11/SEEDSTICK (AGL11/STK) subfamily plays conserved functions in the seed development of flowering plants. Camellia japonica is a world-famous ornamental flower, and its seed kernels are rich in highly valuable fatty acids. Seed abortion has been found to be common in C. japonica, but little is known about how it is regulated during seed development. In this study, we performed a genome-wide analysis of the MADS-box gene the in C. japonica genome and identified 126 MADS-box genes. Through gene expression profiling in various tissue types, we revealed the C/D-class MADS-box genes were preferentially expressed in seed-related tissues. We identified the AGL11/STK-like gene, CjSTK, and showed that it contained a typical STK motif and exclusively expressed during seed development. We found a significant increase in the CjSTK expression level in aborted seeds compared with normally developing seeds. Furthermore, overexpression of CjSTK in Arabidopsis thaliana caused shorter pods and smaller seeds. Taken together, we concluded that the fine regulation of the CjSTK expression at different stages of seed development is critical for ovule formation and seed abortion in C. japonica. The present study provides evidence revealing the regulation of seed development in Camellia.

Common Bean (Phaseolus vulgaris L.) NAC Transcriptional Factor PvNAC52 Enhances Transgenic Arabidopsis Resistance to Salt, Alkali, Osmotic, and ABA Stress by Upregulating Stress-Responsive Genes.

The NAC family of transcription factors includes no apical meristem (NAM), Arabidopsis thaliana transcription activator 1/2 (ATAF1/2), and cup-shaped cotyledon (CUC2) proteins, which are unique to plants, contributing significantly to their adaptation to environmental challenges. In the present study, we observed that the PvNAC52 protein is predominantly expressed in the cell membrane, cytoplasm, and nucleus. Overexpression of PvNAC52 in Arabidopsis strengthened plant resilience to salt, alkali, osmotic, and ABA stresses. PvNAC52 significantly (p < 0.05) reduced the degree of oxidative damage to cell membranes, proline content, and plant water loss by increasing the expression of MSD1, FSD1, CSD1, POD, PRX69, CAT, and P5CS2. Moreover, the expression of genes associated with abiotic stress responses, such as SOS1, P5S1, RD29A, NCED3, ABIs, LEAs, and DREBs, was enhanced by PvNAC52 overexpression. A yeast one-hybrid assay showed that PvNAC52 specifically binds to the cis-acting elements ABRE (abscisic acid-responsive elements, ACGTG) within the promoter. This further suggests that PvNAC52 is responsible for the transcriptional modulation of abiotic stress response genes by identifying the core sequence, ACGTG. These findings provide a theoretical foundation for the further analysis of the targeted cis-acting elements and genes downstream of PvNAC52 in the common bean.

High Overexpression of SiAAP9 Leads to Growth Inhibition and Protein Ectopic Localization in Transgenic Arabidopsis.

Amino acid permeases (AAPs) transporters are crucial for the long-distance transport of amino acids in plants, from source to sink. While Arabidopsis and rice have been extensively studied, research on foxtail millet is limited. This study identified two transcripts of SiAAP9, both of which were induced by NO3- and showed similar expression patterns. The overexpression of SiAAP9L and SiAAP9S in Arabidopsis inhibited plant growth and seed size, although SiAAP9 was found to transport more amino acids into seeds. Furthermore, SiAAP9-OX transgenic Arabidopsis showed increased tolerance to high concentrations of glutamate (Glu) and histidine (His). The high overexpression level of SiAAP9 suggested its protein was not only located on the plasma membrane but potentially on other organelles, as well. Interestingly, sequence deletion reduced SiAAP9's sensitivity to Brefeldin A (BFA), and SiAAP9 had ectopic localization on the endoplasmic reticulum (ER). Protoplast amino acid uptake experiments indicated that SiAAP9 enhanced Glu transport into foxtail millet cells. Overall, the two transcripts of SiAAP9 have similar functions, but SiAAP9L shows a higher colocalization with BFA compartments compared to SiAAP9S. Our research identifies a potential candidate gene for enhancing the nutritional quality of foxtail millet through breeding.

Arabidopsis Transcriptomics Reveals the Role of Lipoxygenase2 (AtLOX2) in Wound-Induced Responses.

In wounded Arabidopsis thaliana leaves, four 13S-lipoxygenases (AtLOX2, AtLOX3, AtLOX4, AtLOX6) act in a hierarchical manner to contribute to the jasmonate burst. This leads to defense responses with LOX2 playing an important role in plant resistance against caterpillar herb-ivory. In this study, we sought to characterize the impact of AtLOX2 on wound-induced phytohormonal and transcriptional responses to foliar mechanical damage using wildtype (WT) and lox2 mutant plants. Compared with WT, the lox2 mutant had higher constitutive levels of the phytohormone salicylic acid (SA) and enhanced expression of SA-responsive genes. This suggests that AtLOX2 may be involved in the biosynthesis of jasmonates that are involved in the antagonism of SA biosynthesis. As expected, the jasmonate burst in response to wounding was dampened in lox2 plants. Generally, 1 h after wounding, genes linked to jasmonate biosynthesis, jasmonate signaling attenuation and abscisic acid-responsive genes, which are primarily involved in wound sealing and healing, were differentially regulated between WT and lox2 mutants. Twelve h after wounding, WT plants showed stronger expression of genes associated with plant protection against insect herbivory. This study highlights the dynamic nature of jasmonate-responsive gene expression and the contribution of AtLOX2 to this pathway and plant resistance against insects.

Identification and Functional Analysis of KH Family Genes Associated with Salt Stress in Rice.

Salinity stress has a great impact on crop growth and productivity and is one of the major factors responsible for crop yield losses. The K-homologous (KH) family proteins play vital roles in regulating plant development and responding to abiotic stress in plants. However, the systematic characterization of the KH family in rice is still lacking. In this study, we performed genome-wide identification and functional analysis of KH family genes and identified a total of 31 KH genes in rice. According to the homologs of KH genes in Arabidopsis thaliana, we constructed a phylogenetic tree with 61 KH genes containing 31 KH genes in Oryza sativa and 30 KH genes in Arabidopsis thaliana and separated them into three major groups. In silico tissue expression analysis showed that the OsKH genes are constitutively expressed. The qRT-PCR results revealed that eight OsKH genes responded strongly to salt stresses, and OsKH12 exhibited the strongest decrease in expression level, which was selected for further study. We generated the Oskh12-knockout mutant via the CRISPR/Cas9 genome-editing method. Further stress treatment and biochemical assays confirmed that Oskh12 mutant was more salt-sensitive than Nip and the expression of several key salt-tolerant genes in Oskh12 was significantly reduced. Taken together, our results shed light on the understanding of the KH family and provide a theoretical basis for future abiotic stress studies in rice.

Genome-Wide Identification of TLP Gene Family in Populus trichocarpa and Functional Characterization of PtTLP6, Preferentially Expressed in Phloem.

Thaumatin-like proteins (TLPs) in plants are involved in diverse biotic and abiotic stresses, including antifungal activity, low temperature, drought, and high salinity. However, the roles of the TLP genes are rarely reported in early flowering. Here, the TLP gene family was identified in P. trichocarpa. The 49 PtTLP genes were classified into 10 clusters, and gene structures, conserved motifs, and expression patterns were analyzed in these PtTLP genes. Among 49 PtTLP genes, the PtTLP6 transcription level is preferentially high in stems, and GUS staining signals were mainly detected in the phloem tissues of the PtTLP6pro::GUS transgenic poplars. We generated transgenic Arabidopsis plants overexpressing the PtTLP6 gene, and its overexpression lines showed early flowering phenotypes. However, the expression levels of main flowering regulating genes were not significantly altered in these PtTLP6-overexpressing plants. Our data further showed that overexpression of the PtTLP6 gene led to a reactive oxygen species (ROS) burst in Arabidopsis, which might advance the development process of transgenic plants. In addition, subcellular localization of PtTLP6-fused green fluorescent protein (GFP) was in peroxisome, as suggested by tobacco leaf transient transformation. Overall, this work provides a comprehensive analysis of the TLP gene family in Populus and an insight into the role of TLPs in woody plants.

Employing Genomic Tools to Explore the Molecular Mechanisms behind the Enhancement of Plant Growth and Stress Resilience Facilitated by a Burkholderia Rhizobacterial Strain.

The rhizobacterial strain BJ3 showed 16S rDNA sequence similarity to species within the Burkholderia genus. Its complete genome sequence revealed a 97% match with Burkholderia contaminans and uncovered gene clusters essential for plant-growth-promoting traits (PGPTs). These clusters include genes responsible for producing indole acetic acid (IAA), osmolytes, non-ribosomal peptides (NRPS), volatile organic compounds (VOCs), siderophores, lipopolysaccharides, hydrolytic enzymes, and spermidine. Additionally, the genome contains genes for nitrogen fixation and phosphate solubilization, as well as a gene encoding 1-aminocyclopropane-1-carboxylate (ACC) deaminase. The treatment with BJ3 enhanced root architecture, boosted vegetative growth, and accelerated early flowering in Arabidopsis. Treated seedlings also showed increased lignin production and antioxidant capabilities, as well as notably increased tolerance to water deficit and high salinity. An RNA-seq transcriptome analysis indicated that BJ3 treatment significantly activated genes related to immunity induction, hormone signaling, and vegetative growth. It specifically activated genes involved in the production of auxin, ethylene, and salicylic acid (SA), as well as genes involved in the synthesis of defense compounds like glucosinolates, camalexin, and terpenoids. The expression of AP2/ERF transcription factors was markedly increased. These findings highlight BJ3's potential to produce various bioactive metabolites and its ability to activate auxin, ethylene, and SA signaling in Arabidopsis, positioning it as a new Burkholderia strain that could significantly improve plant growth, stress resilience, and immune function.

Genome-Wide Identification of CHYR Gene Family in Sophora alopecuroides and Functional Analysis of SaCHYR4 in Response to Abiotic Stress.

Sophora alopecuroides has important uses in medicine, wind breaking, and sand fixation. The CHY-zinc-finger and RING-finger (CHYR) proteins are crucial for plant growth, development, and environmental adaptation; however, genetic data regarding the CHYR family remain scarce. We aimed to investigate the CHYR gene family in S. alopecuroides and its response to abiotic stress, and identified 18 new SaCHYR genes from S. alopecuroides whole-genome data, categorized into 3 subclasses through a phylogenetic analysis. Gene structure, protein domains, and conserved motifs analyses revealed an exon-intron structure and conserved domain similarities. A chromosome localization analysis showed distribution across 12 chromosomes. A promoter analysis revealed abiotic stress-, light-, and hormone-responsive elements. An RNA-sequencing expression pattern analysis revealed positive responses of SaCHYR genes to salt, alkali, and drought stress. SaCHYR4 overexpression considerably enhanced alkali and drought tolerance in Arabidopsis thaliana. These findings shed light on SaCHYR's function and the resistance mechanisms of S. alopecuroides, presenting new genetic resources for crop resistance breeding.

Companion cell mediates wound-stimulated leaf-to-leaf electrical signaling.

Leaf wounding triggers rapid long-range electrical signaling that initiates systemic defense responses to protect the plants from further attack. In Arabidopsis, this process largely depends on clade three GLUTAMATE RECEPTOR-LIKE (GLR) genes GLR3.3 and GLR3.6. In the cellular context, phloem sieve elements and xylem contact cells where GLRs were mostly present are implicated in the signaling events. In spite of that, the spatial requirements of different leaf cell types for leaf-to-leaf signaling remain poorly investigated. In this study, we dissected cell-type-specific long-distance wound signaling mediated by GLR3s and showed that phloem companion cells are critical in shaping the functions of GLR3.3 and GLR3.6 in the signaling pathway. GLR3.3-mediated response is phloem-specific, during which, GLR3.3 has to be renewed from companion cells to allow its function in sieve elements. GLR3.6 functions dually in ectopic phloem companion cells, in addition to xylem contact cells. Furthermore, the action of GLR3.6 in phloem is independent of its paralog GLR3.3 and probably requires synthesis of GLR3.6 from xylem contact cells. Overall, our work highlights that the phloem companion cell is crucial for both GLRs in controlling leaf-to-leaf electrical signaling.

Genome-wide association studies meta-analysis uncovers NOJO and SGS3 novel genes involved in Arabidopsis thaliana primary root development and plasticity.

BACKGROUND: Arabidopsis thaliana primary root growth has become a model for evo-devo studies due to its simplicity and facility to record cell proliferation and differentiation. To identify new genetic components relevant to primary root growth, we used a Genome-Wide Association Studies (GWAS) meta-analysis approach using data published in the last decade. In this work, we performed intra and inter-studies analyses to discover new genetic components that could participate in primary root growth. METHODS AND RESULTS: We used 639 accessions from nine different studies under control conditions and performed different GWAS tests. We found that primary root growth changes were associated with 41 genes, of which six (14.6%) have been previously described as inhibitors or promoters of primary root growth. The knockdown lines of two genes, Suppressor of Gene Silencing (SGS3), involved in tasiRNA processing, and a gene with a Sterile Alpha Motif (SAM) motif named NOJOCH MOOTS (NOJO), confirmed their role as repressors of primary root growth, none has been shown to participate in this developmental process before. CONCLUSIONS: In summary, our GWAS analysis of different available studies identified new genes that participate in primary root growth; two of them were identified as repressors of primary root growth.

Nutrient levels control root growth responses to high ambient temperature in plants.

Global warming will lead to significantly increased temperatures on earth. Plants respond to high ambient temperature with altered developmental and growth programs, termed thermomorphogenesis. Here we show that thermomorphogenesis is conserved in Arabidopsis, soybean, and rice and that it is linked to a decrease in the levels of the two macronutrients nitrogen and phosphorus. We also find that low external levels of these nutrients abolish root growth responses to high ambient temperature. We show that in Arabidopsis, this suppression is due to the function of the transcription factor ELONGATED HYPOCOTYL 5 (HY5) and its transcriptional regulation of the transceptor NITRATE TRANSPORTER 1.1 (NRT1.1). Soybean and Rice homologs of these genes are expressed consistently with a conserved role in regulating temperature responses in a nitrogen and phosphorus level dependent manner. Overall, our data show that root thermomorphogenesis is a conserved feature in species of the two major groups of angiosperms, monocots and dicots, that it leads to a reduction of nutrient levels in the plant, and that it is dependent on environmental nitrogen and phosphorus supply, a regulatory process mediated by the HY5-NRT1.1 module.

XBAT31 regulates reproductive thermotolerance through controlling the accumulation of HSFB2a/B2b under heat stress conditions.

Heat shock transcription factors (HSFs) play a crucial role in heat stress tolerance in vegetative tissues. However, their involvement in reproductive tissues and their post-translational modifications are not well understood. In this study, we identify the E3 ligase XB3 ORTHOLOG 1 IN ARABIDOPSIS THALIANA (XBAT31) as a key player in the ubiquitination and degradation of HSFB2a/B2b. Our results show that the xbat31 mutant exhibits a higher percentage of unfertile siliques and decreased expression of HSPs in flowers under heat stress conditions compared to the wild type. Conversely, the hsfb2a hsfb2b double mutant displays improved reproductive thermotolerance. We find that XBAT31 interacts with HSFB2a/B2b and mediates their ubiquitination. Furthermore, HSFB2a/B2b ubiquitination is reduced in the xbat31-1 mutant, resulting in higher accumulation of HSFB2a/B2b in flowers under heat stress conditions. Overexpression of HSFB2a or HSFB2b leads to an increase in unfertile siliques under heat stress conditions. Thus, our results dissect the important role of the XBAT31-HSFB2a/B2b module in conferring reproductive thermotolerance in plants.