Maturation of arabidopsis seeds is dependent on glutathione biosynthesis within the embryo

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.     

RML1 and RML2, Arabidopsis genes required for cell proliferation at the root tip.

New cells are produced from the meristematic tissues located at the shoot and root tip throughout the life of higher plants. To investigate the genetic mechanism regulating meristematic activity, we isolated and characterized four single-gene, recessive mutants in Arabidopsis thaliana called root meristemless (rml). Complementation tests identified two RML loci; RML1 maps to chromosome IV and RML2 maps to chromosome III. These mutants produce normal embryonic roots that either did not undergo or experienced limited cell division following germination, resulting in primary roots of less than 2.0 mm in length. Mutants can produce lateral and adventitious roots, which can grow to a length comparable to the embryonic root and arrest, indicating that the growth arrest is unrelated to the embryonic dormancy process. Neither the addition of growth regulators to the media nor the removal of shoots can rescue mutant roots from growth arrest, indicating that the mutant phenotype is not caused by a shortage of known growth regulators or by a transmissible shoot inhibitor. Normal cell division ability in mutant embryo, shoot, and callus cells indicates that the RML gene functions are not part of the general cell division processes; rather, they are involved specifically in activating the cell division cycle in the root apical cells.     

Fluridone affects quiescent centre division in the Arabidopsis thaliana root stem cell niche

Plants undergo cell division throughout their life in order to maintain their growth. It is well known that root and shoot tip of plants possess meristems, which contain quiescent cells. Fluridone (1-methyl-3-phenyl-5-(3-trifluoromethyl (phenyl))-4-(1H)-pyridinone) is an established inhibitor of both ABA and carotenoid biosynthesis. However, the other functions of fluridone remain undiscovered. In this report, we provide experimental evidence that fluridone plays a role in the division of the quiescent centre of the Arabidopsis root meristem. This study examined the effects of exogenous fluridone and ABA on the development of the stem cell niche in Arabidopsis root. We show that fluridone promoted the division of stem cells in the quiescent centre, whereas exogenous ABA suppressed quiescent centre division. Furthermore, we established a novel regulatory function for fluridone by demonstrating that it plays an important role in postembryonic development.     

The folylpolyglutamate synthetase plastidial isoform is required for postembryonic root development in Arabidopsis

A recessive Arabidopsis (Arabidopsis thaliana) mutant with short primary roots and root hairs was identified from a forward genetic screen. The disrupted gene in the mutant encoded the plastidial isoform of folylpolyglutamate synthetase (FPGS), previously designated as AtDFB, an enzyme that catalyzes the addition of glutamate residues to the folate molecule to form folylpolyglutamates. The short primary root of atdfb was associated with a disorganized quiescent center, dissipated auxin gradient in the root cap, bundled actin cytoskeleton, and reduced cell division and expansion. The accumulation of monoglutamylated forms of some folate classes in atdfb was consistent with impaired FPGS function. The observed cellular defects in roots of atdfb underscore the essential role of folylpolyglutamates in the highly compartmentalized one-carbon transfer reactions (C1 metabolism) that lead to the biosynthesis of compounds required for metabolically active cells found in the growing root apex. Indeed, metabolic profiling uncovered a depletion of several amino acids and nucleotides in atdfb indicative of broad alterations in metabolism. Methionine and purines, which are synthesized de novo in plastids via C1 enzymatic reactions, were particularly depleted. The root growth and quiescent center defects of atdfb were rescued by exogenous application of 5-formyl-tetrahydrofolate, a stable folate that was readily converted to metabolically active folates. Collectively, our results indicate that AtDFB is the predominant FPGS isoform that generates polyglutamylated folate cofactors to support C1 metabolism required for meristem maintenance and cell expansion during postembryonic root development in Arabidopsis.     

A mechanism coordinating root elongation,endodermal differentiation,redox homeostasis and stress response

Root growth relies on both cell division and cell elongation, which occur in the meristem and elongation zones, respectively. SCARECROW (SCR) and SHORT-ROOT (SHR) are GRAS family genes essential for root growth and radial patterning in the Arabidopsis root. Previous studies showed that SCR and SHR promote root growth by suppressing cytokinin response in the meristem, but there is evidence that SCR expressed beyond the meristem is also required for root growth. Here we report a previously unknown role for SCR in promoting cell elongation. Consistent with this, we found that the scr mutant accumulated a higher level of reactive oxygen species (ROS) in the elongation zone, which is probably due to decreased expression of peroxidase gene 3, which consumes hydrogen peroxide in a reaction leading to Casparian strip formation. When the oxidative stress response was blocked in the scr mutant by mutation in ABSCISIC ACID 2 (ABA2) or when the redox status was ameliorated by the upbeat 1 (upb1) mutant, the root became significantly longer, with longer cells and a larger and more mitotically active meristem. Remarkably, however, the stem cell and radial patterning defects in the double mutants still persisted. Since ROS and peroxidases are essential for endodermal differentiation, these results suggest that SCR plays a role in coordinating cell elongation, endodermal differentiation, redox homeostasis and oxidative stress response in the root. We also provide evidence that this role of SCR is independent of SHR, even though they function similarly in other aspects of root growth and development.     

Cystathionine beta‐lyase is crucial for embryo patterning and the maintenance of root stem cell niche in Arabidopsis

The growth and development of roots in plants depends on the specification and maintenance of the root apical meristem. Here, we report the identification of CBL, a gene required for embryo and root development in Arabidopsis, and encodes cystathionine beta‐lyase (CBL), which catalyzes the penultimate step in methionine (Met) biosynthesis, and which also led to the discovery of a previous unknown, but crucial, metabolic contribution by the Met biosynthesis pathway. CBL is expressed in embryos and shows quiescent center (QC)‐enriched expression pattern in the root. cbl mutant has impaired embryo patterning, defective root stem cell niche, stunted root growth, and reduces accumulation of the root master regulators PLETHORA1 (PLT1) and PLT2. Furthermore, mutation in CBL severely decreases abundance of several PIN‐FORMED (PIN) proteins and impairs auxin‐responsive gene expression in the root tip. cbl seedlings also exhibit global reduction in histone H3 Lys‐4 trimethylation (H3K4me3) and DNA methylation. Importantly, mutation in CBL reduces the abundance of H3K4me3 modification in PLT1/2 genes and downregulates their expression. Overexpression of PLT2 partially rescues cbl root meristem defect, suggesting that CBL acts in part through PLT1/2. Moreover, exogenous supplementation of Met also restores the impaired QC activity and the root growth defects of cbl. Taken together, our results highlight the unique role of CBL to maintain the root stem cell niche by cooperative actions between Met biosynthesis and epigenetic modification of key developmental regulators.     

Arabidopsis ASA1 Is Important for Jasmonate-Mediated Regulation of Auxin Biosynthesis and Transport during Lateral Root Formation

Plant roots show an impressive degree of plasticity in adapting their branching patterns to ever-changing growth conditions. An important mechanism underlying this adaptation ability is the interaction between hormonal and developmental signals. Here, we analyze the interaction of jasmonate with auxin to regulate lateral root (LR) formation through characterization of an Arabidopsis thaliana mutant, jasmonate-induced defective lateral root1 (jdl1/asa1-1). We demonstrate that, whereas exogenous jasmonate promotes LR formation in wild-type plants, it represses LR formation in jdl1/asa1-1. JDL1 encodes the auxin biosynthetic gene ANTHRANILATE SYNTHASE α1 (ASA1), which is required for jasmonate-induced auxin biosynthesis. Jasmonate elevates local auxin accumulation in the basal meristem of wild-type roots but reduces local auxin accumulation in the basal meristem of mutant roots, suggesting that, in addition to activating ASA1-dependent auxin biosynthesis, jasmonate also affects auxin transport. Indeed, jasmonate modifies the expression of auxin transport genes in an ASA1-dependent manner. We further provide evidence showing that the action mechanism of jasmonate to regulate LR formation through ASA1 differs from that of ethylene. Our results highlight the importance of ASA1 in jasmonate-induced auxin biosynthesis and reveal a role for jasmonate in the attenuation of auxin transport in the root and the fine-tuning of local auxin distribution in the root basal meristem.     

Glycerol Affects Root Development through Regulation of Multiple Pathways in Arabidopsis

Glycerol metabolism has been well studied biochemically. However, the means by which glycerol functions in plant development is not well understood. This study aimed to investigate the mechanism underlying the effects of glycerol on root development in Arabidopsis thaliana. Exogenous glycerol inhibited primary root growth and altered lateral root development in wild-type plants. These phenotypes appeared concurrently with increased endogenous glycerol-3-phosphate (G3P) and H₂O₂ contents in seedlings, and decreased phosphate levels in roots. Upon glycerol treatment, G3P level and root development did not change in glycerol kinase mutant gli1, but G3P level increased in gpdhc1 and fad-gpdh mutants, which resulted in more severely impaired root development. Overexpression of the FAD-GPDH gene attenuated the alterations in G3P, phosphate and H₂O₂ levels, leading to increased tolerance to exogenous glycerol, which suggested that FAD-GPDH plays an important role in modulating this response. Free indole-3-acetic acid (IAA) content increased by 46%, and DR5pro::GUS staining increased in the stele cells of the root meristem under glycerol treatment, suggesting that glycerol likely alters normal auxin distribution. Decreases in PIN1 and PIN7 expression, β-glucuronidase (GUS) staining in plants expressing PIN7pro::GUS and green fluorescent protein (GFP) fluorescence in plants expressing PIN7pro::PIN7-GFP were observed, indicating that polar auxin transport in the root was downregulated under glycerol treatment. Analyses with auxin-related mutants showed that TIR1 and ARF7 were involved in regulating root growth under glycerol treatment. Glycerol-treated plants showed significant reductions in root meristem size and cell number as revealed by CYCB1;1pro::GUS staining. Furthermore, the expression of CDKA and CYCB1 decreased significantly in treated plants compared with control plants, implying possible alterations in cell cycle progression. Our data demonstrated that glycerol treatment altered endogenous levels of G3P, phosphate and ROS, affected auxin distribution and cell division in the root meristem, and eventually resulted in modifications of root development.     

The STT3a subunit isoform of the Arabidopsis oligosaccharyltransferase controls adaptive responses to salt/osmotic stress

Arabidopsis stt3a-1 and stt3a-2 mutations cause NaCl/osmotic sensitivity that is characterized by reduced cell division in the root meristem. Sequence comparison of the STT3a gene identified a yeast ortholog, STT3, which encodes an essential subunit of the oligosaccharyltransferase complex that is involved in protein N-glycosylation. NaCl induces the unfolded protein response in the endoplasmic reticulum (ER) and cell cycle arrest in root tip cells of stt3a seedlings, as determined by expression profiling of ER stress-responsive chaperone (BiP-GUS) and cell division (CycB1;1-GUS) genes, respectively. Together, these results indicate that plant salt stress adaptation involves ER stress signal regulation of cell cycle progression. Interestingly, a mutation (stt3b-1) in another Arabidopsis STT3 isogene (STT3b) does not cause NaCl sensitivity. However, the stt3a-1 stt3b-1 double mutation is gametophytic lethal. Apparently, STT3a and STT3b have overlapping and essential functions in plant growth and developmental processes, but the pivotal and specific protein glycosylation that is a necessary for recovery from the unfolded protein response and for cell cycle progression during salt/osmotic stress recovery is associated uniquely with the function of the STT3a isoform.     

Enhancement of secondary xylem cell proliferation by Arabidopsis cyclin D overexpression in tobacco plants

Secondary xylem is composed of daughter cells produced by the vascular cambium in the stem. Cell proliferation of the secondary xylem is the result of long-range cell division in the vascular cambium. Most xylem cells have a thickened secondary cell wall, representing a large amount of biomass storage. Therefore, regulation of cell division in the vascular cambium and differentiation into secondary xylem is important for biomass production. Cell division is regulated by cell cycle regulators. In this study, we confirm that cell cycle regulators influence cell division in the vascular cambium in tobacco. We produced transgenic tobacco that expresses Arabidopsis thaliana cyclin D2;1 (AtcycD2;1) and AtE2Fa-DPa under the control of the CaMV35S promoter. Each gene is a positive regulator of the cell cycle, and is known to influence the transition from G1 phase to S phase. AtcycD2;1-overexpressing tobacco had more secondary xylem cells when compared with control plants. In order to evaluate cell division activity in the vascular cambium, we prepared a Populus trichocarpa cycB1;1 (PtcycB1;1) promoter containing a destruction box motif for ubiquitination and a β-glucuronidase-encoding gene (PtcycB1;1pro:GUS). In transgenic tobacco containing PtcycB1;1pro:GUS, GUS staining was specifically observed in meristem tissues, such as the root apical meristem and vascular cambium. In addition, mitosis-monitoring plants containing AtcycD2;1 had stronger GUS staining in the cambium when compared with control plants. Our results indicated that overexpression of AtcycD enhances cell division in the vascular cambium and increases secondary xylem differentiation in tobacco. Key message We succeeded in inducing cell proliferation of cambium and enlargement of secondary xylem region by AtcycD overexpression. We also evaluated mitotic activity in cambium using cyclin-GUS fusion protein from poplar.     

Gene NANA regulates cell proliferation in Arabidopsis thaliana shoot apical meristem without interaction with CLV1, CLV2, CLV3

A constancy of stem cell pool in shoot apical meristem of Arabidopsis thaliana is provided by a genetic regulation system with negative feedback loop based on the interaction of the gene WUS, which maintains indeterminate state of cells, with CLV genes, which restrict the level of WUS expression and stem cell pool size. clv mutations lead to an increase in the pool of stem cells in the apical and floral meristems and wus mutation leads to the opposite effect. Mutation na (nana), like wus mutation, causes premature termination of shoot apical meristem function, although it does not affect the activity of the flower meristem. To elucidate the role of NA in the control of shoot apical meristem functioning, the interaction of NA with CLV genes were investigated. Additive phenotype of double mutants na clv1, na clv2-1, and na clv3-2 indicates that the NA gene makes an independent contribution to the functioning of the shoot apical meristem. It is assumed that the NA gene controls apical meristem cell proliferation during the transition to the reproductive phase of plant development, acting much later and independently of the genes WUS-CLV.     

Arabidopsis CULLIN3 Genes Regulate Primary Root Growth and Patterning by Ethylene-Dependent and -Independent Mechanisms

CULLIN3 (CUL3) together with BTB-domain proteins form a class of Cullin-RING ubiquitin ligases (called CRL3s) that control the rapid and selective degradation of important regulatory proteins in all eukaryotes. Here, we report that in the model plant Arabidopsis thaliana, CUL3 regulates plant growth and development, not only during embryogenesis but also at post-embryonic stages. First, we show that CUL3 modulates the emission of ethylene, a gaseous plant hormone that is an important growth regulator. A CUL3 hypomorphic mutant accumulates ACS5, the rate-limiting enzyme in ethylene biosynthesis and as a consequence exhibits a constitutive ethylene response. Second, we provide evidence that CUL3 regulates primary root growth by a novel ethylene-dependant pathway. In particular, we show that CUL3 knockdown inhibits primary root growth by reducing root meristem size and cell number. This phenotype is suppressed by ethylene-insensitive or resistant mutations. Finally, we identify a function of CUL3 in distal root patterning, by a mechanism that is independent of ethylene. Thus, our work highlights that CUL3 is essential for the normal division and organisation of the root stem cell niche and columella root cap cells.     

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm⁻¹ h⁻¹) and cell division (cells cell⁻¹ h⁻¹). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement.