Twin Embryos in Arabidopsis thaliana KATANIN 1 Mutants.

Regulation of microtubule dynamics is crucial during key developmental transitions such as gametogenesis, fertilization, embryogenesis, and seed formation, where cells undergo rapid changes in shape and function. In plants, katanin plays an essential role in microtubule dynamics. This study investigates two seed developmental mutants in Arabidopsis thaliana, named elk5-1D (erecta-like 5, ELK5) and loo1 (lollipop 1), which are characterized by round seeds, dwarfism, and fertility defects. Notably, elk5-1D exhibits a dominant inheritance pattern, whereas loo1 is recessive. Through positional cloning, we identified both mutants as new alleles of the KATANIN 1 (KTN1) gene, which encodes a microtubule-severing enzyme critical for cell division and morphology. Mutations in KTN1 disrupt embryo cell division and lead to the emergence of a twin embryo phenotype. Our findings underscore the essential role of KTN1 in fertility and early embryonic development, potentially influencing the fate of reproductive cells.

The receptor kinase SRF3 coordinates iron-level and flagellin dependent defense and growth responses in plants.

Iron is critical for host-pathogen interactions. While pathogens seek to scavenge iron to spread, the host aims at decreasing iron availability to reduce pathogen virulence. Thus, iron sensing and homeostasis are of particular importance to prevent host infection and part of nutritional immunity. While the link between iron homeostasis and immunity pathways is well established in plants, how iron levels are sensed and integrated with immune response pathways remains unknown. Here we report a receptor kinase SRF3, with a role in coordinating root growth, iron homeostasis and immunity pathways via regulation of callose synthases. These processes are modulated by iron levels and rely on SRF3 extracellular and kinase domains which tune its accumulation and partitioning at the cell surface. Mimicking bacterial elicitation with the flagellin peptide flg22 phenocopies SRF3 regulation upon low iron levels and subsequent SRF3-dependent responses. We propose that SRF3 is part of nutritional immunity responses involved in sensing external iron levels.

Antidiabetic and Antihyperlipidemic Effects of Methanolic Extract of Leaves of Spondias mombin in Streptozotocin-Induced Diabetic Rats.

Objective: Spondias mombin is a plant that reported to have anticonvulsant, antimicrobial, antioxidant, antiulcer, antiasthmatic, and wound healing activities. Diabetes dyslipidemic effect of Spondias mombin leaves is not clear. Hence, current study planned to evaluate the antidiabetic and antihyperlipidemic effects of methanolic extract of leaves of Spondias mombin (MESM) in streptozotocin (STZ) induced diabetic rats. Methods: Phytochemicals were determined by standard method and antioxidant activity was determined by DPPH free radical scavenging and FRAP assay. Diabetes was induced by injecting a single dose of STZ (55 mg/kg) into female sprague dawley rats. After 3 days of induction of diabetes, the diabetic animals were treated for 28 days with MESM (125, 250, and 500 mg/kg) and glibenclamide (20 mg/kg) orally. The body weight of rats and blood glucose levels were monitored at regular intervals during the experiment. At the end of study, blood sample was collected from all the animals and subjected to biochemical, lipid profile, and they were sacrificed and their organs such as pancreas, liver and kidney were used for histopathological analysis. Results: Quantitative analysis of MESM showed the presence of anthraquinone, tannins, saponins, steroid, phenols, flavonoids, alkaloids, and reducing sugars. Reduction in body weight and elevated blood glucose were observed in diabetic rats. Treatment with MESM in a concentration of 125, 250, and 500 mg/kg significantly reversed the elevated levels of blood glucose, reduced aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin, urea, creatinine, total serum cholesterol (TC), serum triglyceride (TG), low-density lipoprotein (LDL), Very low-density lipoprotein (VLDL), and increased plasma insulin, total protein, albumin, globulin, A/G ratio, and high-density lipoprotein (HDL). Conclusion: MESM exhibited a significant antidiabetic and antihyperlipidemic activities against STZ-induced diabetes in rats.

The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation.

Gene regulation via N6-methyladenosine (m6A) in mRNA involves RNA-binding proteins that recognize m6A via a YT521-B homology (YTH) domain. The plant YTH domain proteins ECT2 and ECT3 act genetically redundantly in stimulating cell proliferation during organogenesis, but several fundamental questions regarding their mode of action remain unclear. Here, we use HyperTRIBE (targets of RNA-binding proteins identified by editing) to show that most ECT2 and ECT3 targets overlap, with only a few examples of preferential targeting by either of the two proteins. HyperTRIBE in different mutant backgrounds also provides direct views of redundant, ectopic, and specific target interactions of the two proteins. We also show that contrary to conclusions of previous reports, ECT2 does not accumulate in the nucleus. Accordingly, inactivation of ECT2, ECT3, and their surrogate ECT4 does not change patterns of polyadenylation site choice in ECT2/3 target mRNAs, but does lead to lower steady-state accumulation of target mRNAs. In addition, mRNA and microRNA expression profiles show indications of stress response activation in ect2/ect3/ect4 mutants, likely via indirect effects. Thus, previous suggestions of control of alternative polyadenylation by ECT2 are not supported by evidence, and ECT2 and ECT3 act largely redundantly to regulate target mRNA, including its abundance, in the cytoplasm.

Gene expression variation in Arabidopsis embryos at single-nucleus resolution.

Soon after fertilization of egg and sperm, plant genomes become transcriptionally activated and drive a series of coordinated cell divisions to form the basic body plan during embryogenesis. Early embryonic cells rapidly diversify from each other, and investigation of the corresponding gene expression dynamics can help elucidate underlying cellular differentiation programs. However, current plant embryonic transcriptome datasets either lack cell-specific information or have RNA contamination from surrounding non-embryonic tissues. We have coupled fluorescence-activated nuclei sorting together with single-nucleus mRNA-sequencing to construct a gene expression atlas of Arabidopsis thaliana early embryos at single-cell resolution. In addition to characterizing cell-specific transcriptomes, we found evidence that distinct epigenetic and transcriptional regulatory mechanisms operate across emerging embryonic cell types. These datasets and analyses, as well as the approach we devised, are expected to facilitate the discovery of molecular mechanisms underlying pattern formation in plant embryos. This article has an associated 'The people behind the papers' interview.

Green Synthesis and Characterization of Silver Nanoparticles Using Spondias mombin Extract and Their Antimicrobial Activity against Biofilm-Producing Bacteria.

Multidrug resistant bacteria create a challenging situation for society to treat infections. Multidrug resistance (MDR) is the reason for biofilm bacteria to cause chronic infection. Plant-based nanoparticles could be an alternative solution as potential drug candidates against these MDR bacteria, as many plants are well known for their antimicrobial activity against pathogenic microorganisms. Spondias mombin is a traditional plant which has already been used for medicinal purposes as every part of this plant has been proven to have its own medicinal values. In this research, the S. mombin extract was used to synthesise AgNPs. The synthesized AgNPs were characterized and further tested for their antibacterial, reactive oxygen species and cytotoxicity properties. The characterization results showed the synthesized AgNPs to be between 8 to 50 nm with -11.52 of zeta potential value. The existence of the silver element in the AgNPs was confirmed with the peaks obtained in the EDX spectrometry. Significant antibacterial activity was observed against selected biofilm-forming pathogenic bacteria. The cytotoxicity study with A. salina revealed the LC50 of synthesized AgNPs was at 0.81 mg/mL. Based on the ROS quantification, it was suggested that the ROS production, due to the interaction of AgNP with different bacterial cells, causes structural changes of the cell. This proves that the synthesized AgNPs could be an effective drug against multidrug resistant bacteria.

Antagonistic activities of cotranscriptional regulators within an early developmental window set FLC expression level.

Quantitative variation in expression of the Arabidopsis floral repressor FLC influences whether plants overwinter before flowering, or have a rapid cycling habit enabling multiple generations a year. Genetic analysis has identified activators and repressors of FLC expression but how they interact to set expression level is poorly understood. Here, we show that antagonistic functions of the FLC activator FRIGIDA (FRI) and the repressor FCA, at a specific stage of embryo development, determine FLC expression and flowering. FRI antagonizes an FCA-induced proximal polyadenylation to increase FLC expression and delay flowering. Sector analysis shows that FRI activity during the early heart stage of embryo development maximally delays flowering. Opposing functions of cotranscriptional regulators during an early embryonic developmental window thus set FLC expression levels and determine flowering time.

Correction: The AGC protein kinase UNICORN controls planar growth by attenuating PDK1 in Arabidopsis thaliana.

[This corrects the article DOI: 10.1371/journal.pgen.1007927.].

The AGC protein kinase UNICORN controls planar growth by attenuating PDK1 in Arabidopsis thaliana.

Tissue morphogenesis critically depends on the coordination of cellular growth patterns. In plants, many organs consist of clonally distinct cell layers, such as the epidermis, whose cells undergo divisions that are oriented along the plane of the layer. The developmental control of such planar growth is poorly understood. We have previously identified the Arabidopsis AGCVIII-class protein kinase UNICORN (UCN) as a central regulator of this process. Plants lacking UCN activity show spontaneous formation of ectopic multicellular protrusions in integuments and malformed petals indicating that UCN suppresses uncontrolled growth in those tissues. In the current model UCN regulates planar growth of integuments in part by directly repressing the putative transcription factor ABERRANT TESTA SHAPE (ATS). Here we report on the identification of 3-PHOSPHOINOSITIDE-DEPENDENT PROTEIN KINASE 1 (PDK1) as a novel factor involved in UCN-mediated growth control. PDK1 constitutes a basic component of signaling mediated by AGC protein kinases throughout eukaryotes. Arabidopsis PDK1 is implied in stress responses and growth promotion. Here we show that loss-of-function mutations in PDK1 suppress aberrant growth in integuments and petals of ucn mutants. Additional genetic, in vitro, and cell biological data support the view that UCN functions by repressing PDK1. Furthermore, our data indicate that PDK1 is indirectly required for deregulated growth caused by ATS overexpression. Our findings support a model proposing that UCN suppresses ectopic growth in integuments through two independent processes: the attenuation of the protein kinase PDK1 in the cytoplasm and the repression of the transcription factor ATS in the nucleus.

Novel small RNA spike-in oligonucleotides enable absolute normalization of small RNA-Seq data.

Normalization of high-throughput small RNA sequencing (sRNA-Seq) data is required to compare sRNA levels across different samples. Commonly used relative normalization approaches can cause erroneous conclusions due to fluctuating small RNA populations between tissues. We developed a set of sRNA spike-in oligonucleotides (sRNA spike-ins) that enable absolute normalization of sRNA-Seq data across independent experiments, as well as the genome-wide estimation of sRNA:mRNA stoichiometries when used together with mRNA spike-in oligonucleotides.

Microscopic analysis of Arabidopsis ovules.

Ovules are the major female reproductive organs in higher plants. Furthermore, ovules of Arabidopsis thaliana are successfully used as model system to study plant organogenesis. Here we describe two microscopic techniques to analyze ovule development in Arabidopsis. Both methods involve fixed specimens and allow rapid, easy, and reproducible morphological comparisons between wild-type and mutant ovule development.

Genetic analysis of ectopic growth suppression during planar growth of integuments mediated by the Arabidopsis AGC protein kinase UNICORN.

BACKGROUND: The coordination of growth within a tissue layer is of critical importance for tissue morphogenesis. For example, cells within the epidermis undergo stereotypic cell divisions that are oriented along the plane of the layer (planar growth), thereby propagating the layered epidermal structure. Little is known about the developmental control that regulates such planar growth in plants. Recent evidence suggested that the Arabidopsis AGC VIII protein kinase UNICORN (UCN) maintains planar growth by suppressing the formation of ectopic multicellular protrusions in several floral tissues including integuments. In the current model UCN controls this process during integument development by directly interacting with the ABERRANT TESTA SHAPE (ATS) protein, a member of the KANADI (KAN) family of transcription factors, thereby repressing its activity. Here we report on the further characterization of the UCN mechanism. RESULTS: Phenotypic analysis of flowers of ucn-1 plants impaired in floral homeotic gene activity revealed that any of the four floral whorls could produce organs carrying ucn-1 protrusions. The ectopic outgrowths of ucn integuments did not accumulate detectable signals of the auxin and cytokinin reporters DR5rev::GFP and ARR5::GUS, respectively. Furthermore, wild-type and ucn-1 seedlings showed similarly strong callus formation upon in vitro culture on callus-inducing medium. We also show that ovules of ucn-1 plants carrying the dominant ats allele sk21-D exhibited more pronounced protrusion formation. Finally ovules of ucn-1 ett-1 double mutants and ucn-1 ett-1 arf4-1 triple mutants displayed an additive phenotype. CONCLUSIONS: These data deepen the molecular insight into the UCN-mediated control of planar growth during integument development. The presented evidence indicates that UCN downstream signaling does not involve the control of auxin or cytokinin homeostasis. The results also reveal that UCN interacts with ATS independently of an ATS/ETT complex required for integument initiation and they further emphasize the necessity to balance UCN and ATS proteins during maintenance of planar growth in integuments.

Microscopic analysis of ovule development in Arabidopsis thaliana.

Ovules are the major female reproductive organs in higher plants. In addition, ovules of Arabidopsis thaliana are successfully used as model system to study plant organogenesis. Here we describe two microscopic techniques to analyze Arabidopsis ovule development from the organ to the cellular level in a rapid and reproducible fashion. Both methods are of great value when comparing the morphology of wild-type and mutant ovule development.

On the genetic control of planar growth during tissue morphogenesis in plants.

Tissue morphogenesis requires extensive intercellular communication. Plant organs are composites of distinct radial cell layers. A typical layer, such as the epidermis, is propagated by stereotypic anticlinal cell divisions. It is presently unclear what mechanisms coordinate cell divisions relative to the plane of a layer, resulting in planar growth and maintenance of the layer structure. Failure in the regulation of coordinated growth across a tissue may result in spatially restricted abnormal growth and the formation of a tumor-like protrusion. Therefore, one way to approach planar growth control is to look for genetic mutants that exhibit localized tumor-like outgrowths. Interestingly, plants appear to have evolved quite robust genetic mechanisms that govern these aspects of tissue morphogenesis. Here we provide a short summary of the current knowledge about the genetics of tumor formation in plants and relate it to the known control of coordinated cell behavior within a tissue layer. We further portray the integuments of Arabidopsis thaliana as an excellent model system to study the regulation of planar growth. The value of examining this process in integuments was established by the recent identification of the Arabidopsis AGC VIII kinase UNICORN as a novel growth suppressor involved in the regulation of planar growth and the inhibition of localized ectopic growth in integuments and other floral organs. An emerging insight is that misregulation of central determinants of adaxial-abaxial tissue polarity can lead to the formation of spatially restricted multicellular outgrowths in several tissues. Thus, there may exist a link between the mechanisms regulating adaxial-abaxial tissue polarity and planar growth in plants.

Regulation of planar growth by the Arabidopsis AGC protein kinase UNICORN.

The spatial coordination of growth is of central importance for the regulation of plant tissue architecture. Individual layers, such as the epidermis, are clonally propagated and structurally maintained by symmetric cell divisions that are oriented along the plane of the layer. The developmental control of this process is poorly understood. The simple cellular basis and sheet-like structure of Arabidopsis integuments make them an attractive model system to address planar growth. Here we report on the characterization of the Arabidopsis UNICORN (UCN) gene. Analysis of ucn integuments reveals localized distortion of planar growth, eventually resulting in an ectopic multicellular protrusion. In addition, ucn mutants exhibit ectopic growth in filaments and petals, as well as aberrant embryogenesis. We further show that UCN encodes an active AGC VIII kinase. Genetic, biochemical, and cell biological data suggest that UCN suppresses ectopic growth in integuments by directly repressing the KANADI transcription factor ABERRANT TESTA SHAPE. Our findings indicate that UCN represents a unique plant growth regulator that maintains planar growth of integuments by repressing a developmental regulator involved in the control of early integument growth and polarity.

Cytosolic triacylglycerol biosynthetic pathway in oilseeds. Molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase.

Triacylglycerols (TAGs) are the most important storage form of energy for eukaryotic cells. TAG biosynthetic activity was identified in the cytosolic fraction of developing peanut (Arachis hypogaea) cotyledons. This activity was NaF insensitive and acyl-coenzyme A (CoA) dependent. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step in TAG biosynthesis that acylates diacylglycerol to TAG. Soluble DGAT was identified from immature peanuts and purified by conventional column chromatographic procedures. The enzyme has a molecular mass of 41 +/- 1.0 kD. Based on the partial peptide sequence, a degenerate probe was used to obtain the full-length cDNA. The isolated gene shared less than 10% identity with the previously identified DGAT1 and 2 families, but has 13% identity with the bacterial bifunctional wax ester/DGAT. To differentiate the unrelated families, we designate the peanut gene as AhDGAT. Expression of peanut cDNA in Escherichia coli resulted in the formation of labeled TAG and wax ester from [14C]acetate. The recombinant E. coli showed high levels of DGAT activity but no wax ester synthase activity. TAGs were localized in transformed cells with Nile blue A and oil red O staining. The recombinant and native DGAT was specific for 1,2-diacylglycerol and did not utilize hexadecanol, glycerol-3-phosphate, monoacylglycerol, lysophosphatidic acid, and lysophosphatidylcholine. Oleoyl-CoA was the preferred acyl donor as compared to palmitoyl- and stearoyl-CoAs. These data suggest that the cytosol is one of the sites for TAG biosynthesis in oilseeds. The identified pathway may present opportunities of bioengineering oil-yielding plants for increased oil production.