Showing papers in "Plant and Cell Physiology in 2023"

Genome Sequence and Analysis of Nicotiana benthamiana, the Model Plant for Interactions between Organisms

Abstract: Abstract Nicotiana benthamiana is widely used as a model plant for dicotyledonous angiosperms. In fact, the strains used in research are highly susceptible to a wide range of viruses. Accordingly, these strains are subject to plant pathology and plant–microbe interactions. In terms of plant–plant interactions, N. benthamiana is one of the plants that exhibit grafting affinity with plants from different families. Thus, N. benthamiana is a good model for plant biology and has been the subject of genome sequencing analyses for many years. However, N. benthamiana has a complex allopolyploid genome, and its previous reference genome is fragmented into 141,000 scaffolds. As a result, molecular genetic analysis is difficult to perform. To improve this effort, de novo whole-genome assembly was performed in N. benthamiana with Hifi reads, and 1,668 contigs were generated with a total length of 3.1 Gb. The 21 longest scaffolds, regarded as pseudomolecules, contained a 2.8-Gb sequence, occupying 95.6% of the assembled genome. A total of 57,583 high-confidence gene sequences were predicted. Based on a comparison of the genome structures between N. benthamiana and N. tabacum, N. benthamiana was found to have more complex chromosomal rearrangements, reflecting the age of interspecific hybridization. To verify the accuracy of the annotations, the cell wall modification genes involved in grafting were analyzed, which revealed not only the previously indeterminate untranslated region, intron and open reading frame sequences but also the genomic locations of their family genes. Owing to improved genome assembly and annotation, N. benthamiana would increasingly be more widely accessible.

Hyponastic Leaves 1 Interacts with RNA Pol II to Ensure Proper Transcription of MicroRNA Genes

Abstract: Abstract Hyponastic Leaves 1 (HYL1) [also known as Double-stranded RNA-Binding protein 1 (DRB1)] is a double-stranded RNA-binding protein involved in microRNA (miRNA) processing in plants. It is a core component of the Microprocessor complex and enhances the efficiency and precision of miRNA processing by the Dicer-Like 1 protein. In this work, we report a novel function of the HYL1 protein in the transcription of miRNA (MIR) genes. HYL1 colocalizes with RNA polymerase II and affects its distribution along MIR genes. Moreover, proteomic experiments revealed that the HYL1 protein interacts with many transcription factors. Finally, we show that the action of HYL1 is not limited to MIR genes and impacts the expression of many other genes, a majority of which are involved in plastid organization. These discoveries indicate HYL1 as an additional player in gene regulation at the transcriptional level, independent of its role in miRNA biogenesis.

Apoplast-Localized β-Glucosidase Elevates Isoflavone Accumulation in the Soybean Rhizosphere

Abstract: Abstract Plant specialized metabolites (PSMs) are often stored as glycosides within cells and released from the roots with some chemical modifications. While isoflavones are known to function as symbiotic signals with rhizobia and to modulate the soybean rhizosphere microbiome, the underlying mechanisms of root-to-soil delivery are poorly understood. In addition to transporter-mediated secretion, the hydrolysis of isoflavone glycosides in the apoplast by an isoflavone conjugate–hydrolyzing β-glucosidase (ICHG) has been proposed but not yet verified. To clarify the role of ICHG in isoflavone supply to the rhizosphere, we have isolated two independent mutants defective in ICHG activity from a soybean high-density mutant library. In the root apoplastic fraction of ichg mutants, the isoflavone glycoside contents were significantly increased, while isoflavone aglycone contents were decreased, indicating that ICHG hydrolyzes isoflavone glycosides into aglycones in the root apoplast. When grown in a field, the lack of ICHG activity considerably reduced isoflavone aglycone contents in roots and the rhizosphere soil, although the transcriptomes showed no distinct differences between the ichg mutants and wild-types (WTs). Despite the change in isoflavone contents and composition of the root and rhizosphere of the mutants, root and rhizosphere bacterial communities were not distinctive from those of the WTs. Root bacterial communities and nodulation capacities of the ichg mutants did not differ from the WTs under nitrogen-deficient conditions either. Taken together, these results indicate that ICHG elevates the accumulation of isoflavones in the soybean rhizosphere but is not essential for isoflavone-mediated plant–microbe interactions.

Control of Plant Cell Growth and Proliferation by MO25A, a Conserved Major Component of the Mammalian Sterile 20–Like Kinase Pathway

Abstract: Abstract The precise control of cell growth and proliferation underpins the development of plants and animals. These factors affect the development and size of organs and the body. In plants, the growth and proliferation of cells are regulated by environmental stimuli and intrinsic signaling, allowing different cell types to have specific growth and proliferation characteristics. An increasing number of factors that control cell division and growth have been identified. However, the mechanisms underlying cell type–specific cell growth and proliferation characteristics in the normal developmental context are poorly understood. Here, we analyzed the rice mutant osmo25a1, which is defective in the progression of embryogenesis. The osmo25a1 mutant embryo developed incomplete embryonic organs, such as the shoot and root apical meristems. It showed a delayed progression of embryogenesis, associated with the reduced mitotic activity. The causal gene of this mutation encodes a member of the Mouse protein-25A (MO25A) family of proteins that have pivotal functions in a signaling pathway that governs cell proliferation and polarity in animals, yeasts and filamentous fungi. To elucidate the function of plant MO25A at the cellular level, we performed a functional analysis of MO25A in the moss Physcomitrium patens. Physcomitrium patens MO25A was uniformly distributed in the cytoplasm and functioned in cell tip growth and the initiation of cell division in stem cells. Overall, we demonstrated that MO25A proteins are conserved factors that control cell proliferation and growth.

JA-regulated AaGSW1-AaYABBY5/AaWRKY9 complex regulates artemisinin biosynthesis in Artemisia annua.

Abstract: Artemisinin, a sesquiterpene lactone from A. annua, is an essential therapeutic against malaria. YABBY family transcription factor; AaYABBY5 is an activator of AaCYP71AV1 (cytochrome P450-dependent hydroxylase) and AaDBR2 (double bond reductase 2); however, the protein-protein interactions of AaYABBY5, as well as the mechanism of its regulation, are not elucidated before. AaWRKY9 protein is a positive regulator of artemisinin biosynthesis that activates AaGSW1 (Glandular trichome specific WRKY1) and AaDBR2 (double bond reductase 2), respectively. In this study, YABBY-WRKY interactions are revealed to indirectly regulate artemisinin production. AaYABBY5 significantly increased the activity of the luciferase (LUC) gene fused to the promoter of AaGSW1. Towards the molecular basis of this regulation, AaYABBY5 interaction with AaWRKY9 protein was found. The combined effectors AaYABBY5 + AaWRKY9 showed synergistic effects toward the activities of AaGSW1, and AaDBR2 promoters, respectively. In AaYABBY5 over-expression plants, the expression of GSW1 was found significantly increase when compared to that of AaYABBY5 antisense or control plants. Secondly, AaGSW1 was seen as an upstream activator of AaYABBY5. Thirdly, it was found that AaJAZ8, a transcriptional repressor of jasmonates signaling, interacted with AaYABBY5 and attenuated its activity. Co-expression of AaYABBY5 and antiAaJAZ8 in A. annua increased the activity of AaYABBY5 towards artemisinin biosynthesis. For the first time, the current study provided the molecular basis of regulation of artemisinin biosynthesis through YABBY-WRKY interactions and its regulation through AaJAZ8. This knowledge provides AaYABBY5 overexpression plants as a powerful genetic resource for artemisinin biosynthesis.

Cell Cycle Dynamics during Stomatal Development: Window of MUTE Action and Ramification of Its Loss-of-Function on an Uncommitted Precursor

Abstract: Abstract Plants develop in the absence of cell migration. As such, cell division and differentiation need to be coordinated for functional tissue formation. Cellular valves on the plant epidermis, stomata, are generated through a stereotypical sequence of cell division and differentiation events. In Arabidopsis, three master regulatory transcription factors, SPEECHLESS (SPCH), MUTE and FAMA, sequentially drive initiation, proliferation and differentiation of stomata. Among them, MUTE switches the cell cycle mode from proliferative asymmetric division to terminal symmetric division and orchestrates the execution of the single symmetric division event. However, it remains unclear to what extent MUTE regulates the expression of cell cycle genes through the symmetric division and whether MUTE accumulation itself is gated by the cell cycle. Here, we show that MUTE directly upregulates the expression of cell cycle components throughout the terminal cell cycle phases of a stomatal precursor, not only core cell cycle engines but also check-point regulators. Time-lapse live imaging using the multicolor Plant Cell Cycle Indicator revealed that MUTE accumulates up to the early G2 phase, whereas its successor and direct target, FAMA, accumulate at late G2 through terminal mitosis. In the absence of MUTE, meristemoids fail to differentiate and their G1 phase elongates as they reiterate asymmetric divisions. Together, our work provides the framework of cell cycle and master regulatory transcription factors to coordinate a single symmetric cell division and suggests a mechanism for the eventual cell cycle arrest of an uncommitted stem-cell-like precursor at the G1 phase.

Sucrose Signaling Contributes to the Maintenance of Vascular Cambium by Inhibiting Cell Differentiation.

Abstract: Plants produce sugars by photosynthesis and use them for growth and development. Sugars are transported from source-to-sink organs via the phloem in the vasculature. It is well known that vascular development is precisely controlled by plant hormones and peptide hormones. However, the role of sugars in the regulation of vascular development is poorly understood. In this study, we examined the effects of sugars on vascular cell differentiation using a vascular cell induction system named Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL). We found that sucrose has the strongest inhibitory effect on xylem differentiation among several types of sugars. Transcriptome analysis revealed that sucrose suppresses xylem and phloem differentiation from cambial cells. Physiological and genetic analysis suggested that sucrose might function through the BES1 transcription factor, which is the central regulator of vascular cell differentiation. Conditional overexpression of cytosolic invertase led to a decrease in the number of cambium layers due to an imbalance between cell division and differentiation. Taken together, our results suggest that sucrose potentially acts as a signal that integrates environmental conditions with the developmental program.

ASTER-REP, a Database of Asteraceae Sequences for Structural and Functional Studies of Transposable Elements.

Abstract: Journal Article Accepted manuscript ASTER-REP, a Database of Asteraceae Sequences for Structural and Functional Studies of Transposable Elements Get access Maria Ventimiglia, Maria Ventimiglia Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa Italy Search for other works by this author on: Oxford Academic Google Scholar Emanuele Bosi, Emanuele Bosi Department of Earth, Environmental and Life Sciences (DISTAV), University of Genoa, Genova, Corso Europa, 26, 16132 Genova Italy Search for other works by this author on: Oxford Academic Google Scholar Luca Vasarelli, Luca Vasarelli CNR, Istituto di informatica e telematica, Via Giuseppe Moruzzi, 1, 56124 Pisa Italy Search for other works by this author on: Oxford Academic Google Scholar Andrea Cavallini, Andrea Cavallini Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa Italy Search for other works by this author on: Oxford Academic Google Scholar Flavia Mascagni Flavia Mascagni Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa Italy Corresponding author: Flavia Mascagni Email: flavia.mascagni@unipi.it https://orcid.org/0000-0001-9747-8040 Search for other works by this author on: Oxford Academic Google Scholar Plant and Cell Physiology, pcad008, https://doi.org/10.1093/pcp/pcad008 Published: 24 January 2023 Article history Received: 02 January 2023 Revision received: 20 January 2023 Editorial decision: 23 January 2023 Accepted: 23 January 2023 Published: 24 January 2023

Horizontally Acquired Nitrate Reductase Realized Kleptoplastic Photoautotrophy of Rapaza viridis.

Abstract: While photoautotrophic organisms utilize inorganic nitrogen as the nitrogen source, heterotrophic organisms utilize organic nitrogen and thus do not generally have an inorganic nitrogen assimilation pathway. Here we focused on the nitrogen metabolism of Rapaza viridis, a unicellular eukaryote exhibiting kleptoplasty. Although belonging to the lineage of essentially heterotrophic flagellates, R. viridis exploits the photosynthetic products of the kleptoplasts and was therefore suspected to potentially utilize inorganic nitrogen. From the transcriptome data of R. viridis, we identified the gene RvNaRL, which had sequence similarity to nitrate reductases found in plants. Phylogenetic analysis revealed that RvNaRL was acquired by a horizontal gene transfer event. To verify its function of the protein product RvNaRL, we established a RNAi mediated knockdown and a CRISPR-Cas9-mediated knockout experiments for the first time in R. viridis and applied them to this gene. The RvNaRL knockdown and knockout cells exhibited significant growth only when ammonium was supplied. However, in contrast to the wild-type cells, no substantial growth was observed when nitrate was supplied. Such arrested growth in absence of ammonium was attributed to impaired amino acid synthesis due to the deficiency of nitrogen supply from the nitrate assimilation pathway; this in turn resulted in the accumulation of excess photosynthetic products in the form of cytosolic polysaccharide grains as observed. These results indicate that RvNaRL is certainly involved in nitrate assimilation by R. viridis. Thus, we inferred that R. viridis achieved its advanced kleptoplasty for photoautotrophy, owing to acquisition of the nitrate assimilation by the horizontal gene transfer.

Autophagy-Mediated Phosphate Homeostasis in Arabidopsis Involves Modulation of Phosphate Transporters.

Abstract: Autophagy in plants is regulated by diverse signaling cascades in response to environmental changes. Fine-tuning of its activity is critical for the maintenance of cellular homeostasis under basal and stressed conditions. In this study, we compared the Arabidopsis autophagy-related (ATG) system transcriptionally under inorganic phosphate (Pi) deficiency versus nitrogen deficiency and showed that most ATG genes are only moderately upregulated by Pi starvation, with relatively stronger induction of AtATG8f and AtATG8h among the AtATG8 family. We found that Pi shortage increased the formation of GFP-ATG8f-labeled autophagic structures and the autophagic flux in the differential zone of the Arabidopsis root. However, the proteolytic cleavage of GFP-ATG8f and the vacuolar degradation of endogenous ATG8 proteins indicated that Pi limitation does not drastically alter the autophagic flux in the whole roots, implying a cell type-dependent regulation of autophagic activities. At the organismal level, the Arabidopsis atg mutants exhibited decreased shoot Pi concentrations and smaller meristem sizes under Pi sufficiency. Under Pi limitation, these mutants showed enhanced Pi uptake and impaired root cell division and expansion. Despite a reduced steady-state level of several PHOSPHATE TRANSPORTER 1s (PHT1s) in the atg root, cycloheximide treatment analysis suggested that the protein stability of PHT1;1/2/3 is comparable in the Pi-replete wild type and atg5-1. By contrast, the degradation of PHT1;1/2/3 is enhanced in the Pi-deplete atg5-1. Our findings reveal that both basal autophagy and Pi starvation-induced autophagy are required for the maintenance of Pi homeostasis and may modulate the expression of PHT1s through different mechanisms.

OsSYMRK Plays an Essential Role in AM Symbiosis in Rice (Oryza sativa).

Abstract: Arbuscular mycorrhizal (AM) fungi establish mutualistic symbiosis with a wide range of terrestrial plants, including rice. However, the mechanisms underlying the initiation of AM symbiosis have yet to be elucidated, particularly in nonleguminous plants. We previously demonstrated that OsCERK1, a lysin motif (LysM) receptor-like kinase essential for chitin-triggered immunity, also plays a key role in AM symbiosis in rice. However, the mechanisms underlying the regulation of switching between immunity and symbiosis by OsCERK1 have yet to be fully elucidated. SYMRK/DMI2 is a leucine-rich repeat receptor-like kinase associated with both root nodule symbiosis and AM symbiosis in legumes. The homologue of SYMRK in rice, OsSYMRK, has a shorter form than in legumes because OsSYMRK lacks a malectin-like domain (MLD). The MLD reportedly contributes to symbiosis in Lotus japonicus; however, the contribution of OsSYMRK to AM symbiosis in rice remains unclear. Phylogenetic analyses indicated that the MLD of SYMRK/DMI2 is widely conserved even in mosses and ferns but absent in commelinids, including rice. To understand the function of OsSYMRK, we produced an Ossymrk knockout mutant using CRISPR/Cas9 technology. AM colonization was mostly abolished in Ossymrk with a more severe phenotype than Oscerk1. Ca2+ spiking against chitin tetramer was also diminished in Ossymrk. In contrast, comparable defense responses against chitin heptamer to wild type were observed in Ossymrk. Bimolecular fluorescence complementation studies demonstrating an interaction between OsSYMRK and OsCERK1 indicate OsSYMRK may play an important role in switching from immunity to symbiosis through the interaction with OsCERK1 in rice.

The mutual inhibition between PLETHORAs and ARABIDOPSIS RESPONSE REGULATORs controls root zonation.

Abstract: During organogenesis a key step towards the development of a functional organ is the separation of cells in specific domains with different activities. Mutual inhibition of gene expression has been shown to be sufficient to establish and maintain these domains during organogenesis of several multicellular organisms. Here we show that the mutual inhibition between the PLTs and the ARRs transcription factors is sufficient to separate cell division and cell differentiation during root organogenesis. In particular, we show that ARR1 suppresses PLTs activities and that PLTs suppress ARR1 and ARR12 by targeting their protein for degradation via the KMD2 F-box protein. These findings reveal new important aspects of the complex process of root zonation and development.

BBX24 interacts with JAZ3 to promote growth by reducing DELLA activity in shade avoidance.

Abstract: Shade avoidance syndrome (SAS) is a strategy of major adaptive significance and typically includes elongation of the stem and petioles, leaf hyponasty, reduced branching, and phototropic orientation of the plant shoot toward canopy gaps. Cryptochrome 1 and phytochrome B (phyB) are both the major photoreceptors that sense the reduction in blue light fluence rate and the low Red:Far-Red (R:FR) ratio, respectively, both light signals associated with plant density and the resource reallocation when SAS responses are triggered. BBX24 has been implicated in the SAS as a regulator of DELLA activity, but this interaction does not explain all the observed BBX24-dependent regulation in shade light. Here, through a combination of transcriptional meta-analysis and large-scale identification of BBX24-interacting transcription factors, we found that JAZ3, a jasmonic acid signaling component, is a direct target of BBX24. Furthermore, we demonstrated that joint loss of BBX24 and JAZ3 function causes insensitivity to DELLA accumulation, and the defective shade-induced elongation in this mutant is rescued by loss of DELLA or phytochrome B functions. Therefore, we propose that JAZ3 is part of the regulatory network that controls the plant growth in response to shade, through a mechanism in which BBX24 and JAZ3 jointly regulate DELLA activity. Our results provide new insights into the participation of BBX24 and JA signaling in the hypocotyl shade avoidance response in Arabidopsis.

Dissecting the Biology of Rafflesia Species: Current Progress and Future Directions Made Possible with High-Throughput Sequencing Data.

Abstract: The angiosperm Rafflesia exhibits a unique biology, including a growth strategy that involves endophytic parasitism of a specific host, with only the gigantic flower externally visible. The Rafflesia possesses many unique evolutionary, developmental, and morphological features that are rooted in yet to be explained physiological processes. Although studies on the molecular biology of Rafflesia are limited by sampling difficulties due to its rarity in the wild and the short life span of its flower, current advances in high-throughput sequencing technology have allowed for the genome and transcriptome level dissection of the molecular mechanisms behind the unique characteristics of this parasitic plant. In this review, we summarize major findings on the cryptic biology of Rafflesia and provide insights into future research directions. The wealth of data obtained can improve our understanding of Rafflesia species and contribute towards the conservation strategy of this endangered plant.

Exploring novel polytubey reproduction pathways utilizing cumulative genetic tools.

Abstract: In the anthers and ovaries of flowers, pollen grains and embryo sacs are produced with uniform cell compositions. This stable gametogenesis enables elaborate interactions between male and female gametophytes after pollination, forming the highly successful sexual reproduction system in flowering plants. As most ovules are fertilized with a single pollen tube, the resulting genome set in the embryo and endosperm is determined in a single pattern by independent fertilization of the egg cell and central cell by two sperm cells. However, if ovules receive four sperm cells from two pollen tubes, the expected options for genome sets in the developing seeds would more than double. In wild-type Arabidopsis thaliana plants, around 5% of ovules receive two pollen tubes. Recent studies have elucidated the abnormal fertilization in supernumerary pollen tubes and sperm cells related to polytubey, polyspermy, heterofertilization, and fertilization recovery. Analyses of model plants have begun to uncover the mechanisms underlying this new pollen tube biology. Here, we review unusual fertilization phenomena and propose several breeding applications for flowering plants. These arguments contribute to the remodeling of plant reproduction, a challenging concept that alters typical plant fertilization by utilizing the current genetic toolbox.

Effects of Post Anthesis High Temperature Stress on Carbon Partitioning and Starch Biosynthesis in a Spring Wheat (Triticum aestivum L.) Adapted to Moderate Growth Temperatures.

Abstract: This study investigates carbon partitioning in the developing endosperm of a European variety of spring wheat subjected to moderately elevated daytime temperatures (27°C/16°C day/night) from anthesis to grain maturity. Elevated daytime temperatures caused significant reductions in both fresh and dry weights and reduced starch content of harvested grains compared to plants grown under a 20°C/16°C day/night regime. Accelerated grain development caused by elevated temperatures was accounted for by representing plant development as thermal time (°CDPA). We examined effects of high temperature stress (HTS) on uptake and partitioning of [U-14C]-sucrose supplied to isolated endosperms. HTS caused reduced sucrose uptake into developing endosperms from the second major grain filling stage (approximately 260°CDPA) up to maturity. Enzymes involved in sucrose metabolism were unaffected by HTS, whereas key enzyme activities involved in endosperm starch deposition such as ADP-glucose pyrophosphorylase and soluble isoforms of starch synthase were sensitive to HTS throughout grain development. HTS caused a decrease in other major carbon sinks such as evolved CO2, ethanol-soluble material, cell walls and protein. Despite reductions in labelling of carbon pools caused by HTS, the relative proportions of sucrose taken up by endosperm cells allocated to each cellular pool remain unchanged, except for evolved CO2, which increased under HTS and may reflect enhanced respiratory activity. The results of this study show that moderate temperature increases in some temperate wheat cultivars can cause significant yield reductions chiefly through three effects: reduced sucrose uptake by the endosperm, reduced starch synthesis, and increased partitioning of carbon into evolved CO2.

Evolution of lipochitooligosaccharide binding to a LysM-RLK for nodulation in Medicago truncatula.

Abstract: Lysin motif receptor like kinases (LysM-RLKs) are involved in the perception of chitooligosaccharides (COs) and related lipochitooligosaccharides (LCOs) in plants. Expansion and divergence of the gene family during evolution have led to various roles in symbiosis and defence. By studying proteins of the LYR-IA subclass of LysM-RLKs of the Poaceae, we show here that they are high affinity LCO binding proteins with a lower affinity for COs, consistent with a role in LCO perception to establish arbuscular mycorrhiza (AM). In Papilionoid legumes whole genome duplication has resulted in two LYR-IA paralogs, MtLYR1 and MtNFP in Medicago truncatula, with MtNFP playing an essential role in the root nodule symbiosis with nitrogen-fixing rhizobia. We show that MtLYR1 has retained the ancestral LCO binding characteristic and is dispensable for AM. Domain swapping between the three Lysin motifs (LysMs) of MtNFP and MtLYR1 and mutagenesis in MtLYR1 suggest that the MtLYR1 LCO binding site is on the second LysM, and that divergence in MtNFP led to better nodulation, but surprisingly with decreased LCO binding. These results suggest that divergence of the LCO binding site has been important for the evolution of a role of MtNFP in nodulation with rhizobia.

Editor's Note: The Impact of Tobamovirus Infection on Root Development Involves Induction of Auxin Response Factor 10a in Tomato.

Abstract: Journal Article Editor’s Note: The Impact of Tobamovirus Infection on Root Development Involves Induction of Auxin Response Factor 10a in Tomato Get access Plant and Cell Physiology, Volume 63, Issue 12, December 2022, Page 2045, https://doi.org/10.1093/pcp/pcac169 Published: 10 January 2023 Article history Received: 05 December 2022 Corrected and typeset: 10 January 2023 Published: 10 January 2023

Root PRR7 Improves the Accuracy of the Shoot Circadian Clock through Nutrient Transport

Abstract: Abstract The circadian clock allows plants to anticipate and adapt to periodic environmental changes. Organ- and tissue-specific properties of the circadian clock and shoot-to-root circadian signaling have been reported. While this long-distance signaling is thought to coordinate physiological functions across tissues, little is known about the feedback regulation of the root clock on the shoot clock in the hierarchical circadian network. Here, we show that the plant circadian clock conveys circadian information between shoots and roots through sucrose and K+. We also demonstrate that K+ transport from roots suppresses the variance of period length in shoots and then improves the accuracy of the shoot circadian clock. Sucrose measurements and qPCR showed that root sucrose accumulation was regulated by the circadian clock. Furthermore, root circadian clock genes, including PSEUDO-RESPONSE REGULATOR7 (PRR7), were regulated by sucrose, suggesting the involvement of sucrose from the shoot in the regulation of root clock gene expression. Therefore, we performed time-series measurements of xylem sap and micrografting experiments using prr7 mutants and showed that root PRR7 regulates K+ transport and suppresses variance of period length in the shoot. Our modeling analysis supports the idea that root-to-shoot signaling contributes to the precision of the shoot circadian clock. We performed micrografting experiments that illustrated how root PRR7 plays key roles in maintaining the accuracy of shoot circadian rhythms. We thus present a novel directional signaling pathway for circadian information from roots to shoots and propose that plants modulate physiological events in a timely manner through various timekeeping mechanisms.

Root-Expressed Rice PAP3b Enhances Secreted APase Activity and Helps Utilize Organic Phosphate.

Abstract: Phosphate (Pi) deficiency leads to the induction of purple acid phosphatases (PAPs) in plants, which dephosphorylates organic phosphorus complexes in the rhizosphere and intracellular compartments to release Pi. In this study, we demonstrate that OsPAP3b belongs to group III low molecular weight PAP, and is low Pi responsive, preferentially in roots. The expression of OsPAP3b is negatively regulated with Pi re-supply. Interestingly, OsPAP3b was found to be dual localized to the nucleus and secretome. Furthermore, OsPAP3b is transcriptionally regulated by OsPHR2 as substantiated by DNA-protein binding assay. Through in-vitro biochemical assays, we further demonstrate that OsPAP3b is a functional acid phosphatase with broad substrate specificity. Overexpression of OsPAP3b in rice led to increased secreted APase activity and improved mineralization of organic P sources, reflected in better growth of transgenics compared to wild type when grown on organic P as exogenous P substrate. Under Pi deprivation, OsPAP3b knockdown and knockout lines showed no significant changes in total P content and dry biomass. However, the expression of other phosphate starvation-induced (PSI) genes and the levels of metabolites were found to be altered in the overexpression and knockdown lines. In addition, in-vitro pull-down assay revealed multiple putative interacting proteins of OsPAP3b. Our data collectively suggest that OsPAP3b can aid in organic P utilization in rice. The APase isoforms behavior and nuclear localization indicate its additional role, possibly in stress signaling. Considering its important roles, OsPAP3b could be a potential target for improving low Pi adaptation in rice.

A Stereoselective Strigolactone Biosynthesis Catalyzed by a 2-Oxoglutarate-Dependent Dioxygenase in Sorghum.

Abstract: Seeds of root parasitic plants, Striga, Orobanche and Phelipanche spp., are induced to germinate by strigolactones exudated from host roots. In Striga-resistance cultivars of Sorghum bicolor, the loss-of-function of the Low Germination Stimulant 1 (LGS1) gene changes the major strigolactone from 5-deoxystrigol to orobanchol having the opposite C-ring stereochemistry. However, the biosynthetic pathway of 5-deoxystrigol catalyzed by LGS1 has not been fully elucidated. Since the other unknown regulator in addition to LGS1 encoding a sulfotransferase appeared to be necessary for the stereoselective biosynthesis of 5-deoxystrigol, we examined Sobic.005G213500 (Sb3500) encoding a 2-oxoglutarate-dependent dioxygenase as a candidate, which is co-expressed with LGS1 and located on the 5'-upstream of LGS1 in the sorghum genome. When LGS1 was expressed with known strigolactone biosynthetic enzyme genes including the cytochrome P450 SbMAX1a but not Sb3500 in Nicotiana benthamiana leaves, 5-deoxystrigol and its diastereomer 4-deoxyorobanchol were produced in approximately equal amounts, while the production of 5-deoxystrigol was significantly larger than that of 4-deoxyorobanchol when Sb3500 was co-expressed in addition to them. We also confirmed the stereoselective 5-deoxystrigol production by an in vitro feeding experiment using synthetic chemicals with recombinant proteins expressed in E. coli and yeast. This finding demonstrated that Sb3500 is a stereoselective regulator in the conversion of the strigolactone precursor carlactone to 5-deoxystrigol catalyzed by LGS1 and SbMAX1a, providing a detailed understanding of how different strigolactones are produced to combat parasitic weed infestations.

Nucleotide limitation results in impaired photosynthesis, reduced growth and seed yield together with massively altered gene expression

Abstract: Abstract Nucleotide limitation and imbalance is a well-described phenomenon in animal research but understudied in the plant field. A peculiarity of pyrimidine de novo synthesis in plants is the complex subcellular organization. Here, we studied two organellar localized enzymes in the pathway, with chloroplast aspartate transcarbamoylase (ATC) and mitochondrial dihydroorotate dehydrogenase (DHODH). ATC knock-downs were most severely affected, exhibiting low levels of pyrimidine nucleotides, a low energy state, reduced photosynthetic capacity and accumulation of reactive oxygen species. Furthermore, altered leaf morphology and chloroplast ultrastructure were observed in ATC mutants. Although less affected, DHODH knock-down mutants showed impaired seed germination and altered mitochondrial ultrastructure. Thus, DHODH might not only be regulated by respiration but also exert a regulatory function on this process. Transcriptome analysis of an ATC-amiRNA line revealed massive alterations in gene expression with central metabolic pathways being downregulated and stress response and RNA-related pathways being upregulated. In addition, genes involved in central carbon metabolism, intracellular transport and respiration were markedly downregulated in ATC mutants, being most likely responsible for the observed impaired growth. We conclude that impairment of the first committed step in pyrimidine metabolism, catalyzed by ATC, leads to nucleotide limitation and by this has far-reaching consequences on metabolism and gene expression. DHODH might closely interact with mitochondrial respiration, as seen in delayed germination, which is the reason for its localization in this organelle.

A Polyphenol Oxidase Catalyzes Aurone Synthesis in Marchantia polymorpha.

Abstract: Aurones constitute one of the major classes of flavonoids, with a characteristic furanone structure that acts as the C-ring of flavonoids. Members of various enzyme families are involved in aurone biosynthesis in different higher plants, suggesting that, during evolution, plants acquired the ability to biosynthesize aurones independently and convergently. Bryophytes also produce aurones, but the biosynthetic pathways and enzymes involved have not been determined. The present study describes the identification and characterization of a polyphenol oxidase that acts as an aureusidin synthase (MpAS1) in the model liverwort, Marchantia polymorpha. Crude enzyme assays using an M. polymorpha line overexpressing MpMYB14 with high accumulation of aureusidin showed that aureusidin was biosynthesized from naringenin chalcone and converted to riccionidin A. This activity was inhibited by N-phenylthiourea, an inhibitor specific to enzymes of the polyphenol oxidase family. Of the six polyphenol oxidases highly induced in the line overexpressing MpMyb14, one, MpAS1 was found to biosynthesize aureusidin from naringenin chalcone when expressed in Saccharomyces cerevisiae. MpAS1 also recognized eriodictyol chalcone, isoliquiritigenin and butein, showing the highest activity for eriodictyol chalcone. Members of the polyphenol oxidase family in M. polymorpha evolved independently from polyphenol oxidases in higher plants, indicating that aureusidin synthases evolved in parallel in land plants.

Transcriptome Revealed GhPP2C43-A Negatively Regulates Salinity Tolerance in an Introgression Line from a Semi-wild Upland Cotton.

Abstract: Salt damage is one of the major threats to sustainable cotton production owing to the limited arable land in China mainly occupied by the production of staple food crops. Salt-stress tolerant cotton varieties are lacking in production and, the mechanisms underpinning salt-stress tolerance in cotton remain enigmatic. Here, DM37, an intraspecific introgression line from G. hirsutum race yucatanense acc TX-1046 into the G. hirsutum acc TM-1 background, was found to be highly tolerant to salt stress. Its seed germination rate and germination potential were significantly higher than the recipient TM-1 under salt stress. Physiological analysis showed DM37 had higher proline content and Peroxidase activity, as well as lower Na+/K+ ratios at the seedling stage, consistent with higher seedling survival rate after durable salt stress. Furthermore, comparative transcriptome analysis revealed that responsive patterns to salt stress in DM37 were different from TM-1. Weighted Correlation Network Analysis (WGCNA) demonstrated that co-expression modules associated with salt stress in DM37 also differed from TM-1. Out of them, GhPP2C43-A, a phosphatase gene, exhibited negative regulation of salt-stress tolerance verified by VIGS and transgenic Arabidopsis. Gene expression showed GhPP2C43-A in TM-1 was induced by durable salt stress but not in DM37 probably attributing to the variation of cis-element in its promoter, thereby being conferred different salt-stress tolerance. Our result would provide new genes/germplasms from semi-wild cotton in salt-stress tolerant cotton breeding. This study would give us new insights into the mechanisms underpinning the salt-stress tolerance in cotton.