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A coumaroyl-ester-3-hydroxylase insertion mutant reveals the existence of nonredundant meta-hydroxylation pathways and essential roles for phenolic precursors in cell expansion and plant growth.

Cytochromes P450 monooxygenases from the CYP98 family catalyze the meta-hydroxylation step in the phenylpropanoid biosynthetic pathway. The ref8 Arabidopsis (Arabidopsis thaliana) mutant, with a point mutation in the CYP98A3 gene, was previously described to show developmental defects, changes in lignin composition, and lack of soluble sinapoyl esters. We isolated a T-DNA insertion mutant in CYP98A3 and show that this mutation leads to a more drastic inhibition of plant development and inhibition of cell growth. Similar to the ref8 mutant, the insertion mutant has reduced lignin content, with stem lignin essentially made of p-hydroxyphenyl units and trace amounts of guaiacyl and syringyl units. However, its roots display an ectopic lignification and a substantial proportion of guaiacyl and syringyl units, suggesting the occurrence of an alternative CYP98A3-independent meta-hydroxylation mechanism active mainly in the roots. Relative to the control, mutant plantlets produce very low amounts of sinapoyl esters, but accumulate flavonol glycosides. Reduced cell growth seems correlated with alterations in the abundance of cell wall polysaccharides, in particular decrease in crystalline cellulose, and profound modifications in gene expression and homeostasis reminiscent of a stress response. CYP98A3 thus constitutes a critical bottleneck in the phenylpropanoid pathway and in the synthesis of compounds controlling plant development. CYP98A3 cosuppressed lines show a gradation of developmental defects and changes in lignin content (40% reduction) and structure (prominent frequency of p-hydroxyphenyl units), but content in foliar sinapoyl esters is similar to the control. The purple coloration of their leaves is correlated to the accumulation of sinapoylated anthocyanins.

Arabidopsis↗

The ENHANCER OF TRY AND CPC1 gene acts redundantly with TRIPTYCHON and CAPRICE in trichome and root hair cell patterning in Arabidopsis.

The development of trichomes and root hairs in Arabidopsis provide useful models for the study of cell fate determination in plants. A common network of putative transcriptional regulators, including the small MYB proteins TRIPTYCHON (TRY) and CAPRICE (CPC), is known to influence the patterning of both cell types. Here, we used an activation tagging strategy to identify a new regulator, ENHANCER OF TRY AND CPC 1 (ETC1). The ETC1 sequence is similar to TRY and CPC, and ETC1 overexpression causes a reduction in trichome formation and excessive root hair production. The etc1 single mutant has no significant phenotype, but it enhances the effect of cpc and try on trichome and root hair development, which shows that ETC1 function is partially redundant with TRY and CPC. In addition, the etc1 try cpc triple mutant has novel phenotypes, revealing previously unrecognized roles for these regulators in epidermis development. An ETC1 promoter-reporter gene fusion is expressed in the developing trichome and non-hair cells, similar to the expression of TRY and CPC. These results suggest that ETC1, TRY, and CPC act in concert to repress the trichome cell fate in the shoot epidermis and the non-hair cell fate in the root epidermis.

Amino Acid Sequence↗

Ammonium inhibition of Arabidopsis root growth can be reversed by potassium and by auxin resistance mutations aux1, axr1, and axr2.

A novel effect of ammonium ions on root growth was investigated to understand how environmental signals affect organ development. Ammonium ions (3-12 mM) were found to dramatically inhibit Arabidopsis thaliana seedling root growth in the absence of potassium even if nitrate was present. This inhibition could be reversed by including in the growth medium low levels (20-100 microM) of potassium or alkali ions Rb+ and Cs+ but not alkali ions Na+ and Li+. The protective effect of low concentrations of potassium is not due to an inhibition of ammonium uptake. Ammonium inhibition is reversible, because root growth was restored in ammonium-treated seedlings if they were subsequently transferred to medium containing potassium. It is known that plant hormones can inhibit root growth. We found that mutants of Arabidopsis resistant to high levels of auxin and other hormones (aux1, axr1, and axr2) are also resistant to the ammonium inhibition and produce roots in the absence of potassium. Thus, the mechanisms that mediate the ammonium inhibition of root development are linked to hormone metabolic or signaling pathways. These findings have important implications for understanding how environmental signals, especially mineral nutrients, affect plant root development.

Ammonia↗

Nitrate Utilization by Nitrate Reductase-deficient Barley Mutants.

Two nitrate reductase-deficient barley mutants were studied for growth on nitrate and ammonium sources of nitrogen and for resistance to chlorate. Although nitrate reductase-deficient mutants in some species are chlorate-resistant (unable to reduce chlorate to chlorite), the barley mutants used in these studies when grown on nitrate and treated with chlorate were only slightly more resistant to chlorate than the control. When grown to maturity on vermiculite supplemented with either nitrate or ammonium nutrient solutions, the mutants produced as much dry weight and reduced nitrogen per plant as the control. The in vivo and in vitro nitrate reductase activities in the roots and shoots of the mutants grown on nitrate were consistently less than 10% of the control. To avoid the possibility that the mutants received reduced nitrogen from microbial sources, excised embryos were cultured under sterile conditions. Again the mutants were capable of growth and reduced nitrogen accumulation with nitrate as the sole source of nitrogen. In spite of the low apparent nitrate reductase activity, the nitrate reductase-deficient mutants are capable of substantial nitrate reduction.

Journal Article↗

Disruption of RCI2A leads to over-accumulation of Na+ and increased salt sensitivity in Arabidopsis thaliana plants.

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na+ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na+ ions in the cell and shows increased Na+ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na+ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na+ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na+ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO3, Na2SO4, KCl, KNO3, K2SO4 or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl2, MgCl2, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na+ and K+ ions in plants, and contributes to salt tolerance.

Arabidopsis↗

Nodulation inhibition by Rhizobium leguminosarum multicopy nodABC genes and analysis of early stages of plant infection.

During analysis of early events in the infection and nodulation of Vicia hirsuta roots inoculated with normal and mutant strains of Rhizobium leguminosarum and strains containing cloned nodulation (nod) genes, a number of novel observations were made. (i) Alternating zones of curled and straight root hairs were seen on roots of V. hirsuta inoculated with the wild-type strain of R. leguminosarum. This phasing of root hair curling was not seen if plants were grown under continuous light or continuous dark conditions. (ii) Reduced nodulation and delayed nodule initiation was observed with a strain carrying a Tn5 mutation in the nodE gene. In addition the phased root hair curling was absent, and root hair curling was observed along the length of the root. (iii) The nodABC genes cloned on a multicopy plasmid in a wild-type strain inhibited nodulation but induced a continuous root hair curling response. Those few nodules that eventually formed were found to contain bacteria which had lost the plasmid carrying the nodABC genes. (iv) With a strain of Rhizobium cured of its indigenous symbiotic plasmid, but containing the cloned nodABCDEF genes, continuous root hair curling on V. hirsuta was observed. However, no infection threads were observed, and surprisingly, it did appear that initial stages of nodule development occurred. Observations of thin sections of these early developing nodules indicated that early nodule meristematic divisions may have occurred but that no bacteria were found within the nodules and no infection threads were observed either within the nodule bumps or within any of the root hairs. It was concluded that for normal infections to occur, precise regulation of the nod genes is required and that overexpression of the root hair curling genes inhibits the normal infection process.

Bacterial Proteins↗

BMPR1A signaling is necessary for hair follicle cycling and hair shaft differentiation in mice.

Interactions between ectodermal and mesenchymal extracellular signaling pathways regulate hair follicle (HF) morphogenesis and hair cycling. Bone morphogenetic proteins (BMPs) are known to be important in hair follicle development by affecting the local cell fate modulation. To study the role of BMP signaling in the HF, we disrupted Bmpr1a, which encodes the BMP receptor type IA (BMPR1A) in an HF cell-specific manner, using the Cre/loxP system. We found that the differentiation of inner root sheath, but not outer root sheath, was severely impaired in mutant mice. The number of HFs was reduced in the dermis and subcutaneous tissue, and cycling epithelial cells were reduced in mutant mice HFs. Our results strongly suggest that BMPR1A signaling is essential for inner root sheath differentiation and is indispensable for HF renewal in adult skin.

Animals↗

MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for arabidopsis lateral root development.

Although several plant microRNAs (miRNAs) have been shown to play a role in plant development, no phenotype has yet been associated with a reduction or loss of expression of any plant miRNA. Arabidopsis thaliana miR164 was predicted to target five NAM/ATAF/CUC (NAC) domain-encoding mRNAs, including NAC1, which transduces auxin signals for lateral root emergence. Here, we show that miR164 guides the cleavage of endogenous and transgenic NAC1 mRNA, producing 3'-specific fragments. Cleavage was blocked by NAC1 mutations that disrupt base pairing with miR164. Compared with wild-type plants, Arabidopsis mir164a and mir164b mutant plants expressed less miR164 and more NAC1 mRNA and produced more lateral roots. These mutant phenotypes can be complemented by expression of the appropriate MIR164a and MIR164b genomic sequences. By contrast, inducible expression of miR164 in wild-type plants led to decreased NAC1 mRNA levels and reduced lateral root emergence. Auxin induction of miR164 was mirrored by an increase in the NAC1 mRNA 3' fragment, which was not observed in the auxin-insensitive mutants auxin resistant1 (axr1-12), axr2-1, and transport inhibitor response1. Moreover, the cleavage-resistant form of NAC1 mRNA was unaffected by auxin treatment. Our results indicate that auxin induction of miR164 provides a homeostatic mechanism to clear NAC1 mRNA to downregulate auxin signals.

Arabidopsis↗

Identification of dominant mutations that confer increased aluminium tolerance through mutagenesis of the Al-sensitive Arabidopsis mutant, als3-1.

Aluminium (Al) toxicity is a global agricultural problem that occurs in acid soil environments and severely limits root growth and crop productivity. The isolation and characterization of a gene, ALS3, which is absolutely required by Arabidopsis seedlings for growth in an Al-toxic environment was reported previously. Since the als3-1 loss-of-function mutant has extreme root growth inhibition even in the presence of very low levels of Al, it was an excellent candidate for using a mutagenesis approach to identify suppressor mutations that would increase either Al resistance or tolerance in Arabidopsis roots. EMS-mutagenized als3-1 seedlings were screened for mutants that could sustain root growth in an Al-containing environment that is highly toxic to als3-1 but not Col-0 wt. This approach resulted in identification of 12 strong suppressor mutants that reversed the als3-1 phenotype and grew as well or better than Col-0 wt in the presence of high levels of Al. Subsequent analysis of three representative suppressor mutants revealed that the phenotype of each probably arises from dominant gain-of-function mutations at the same locus. Detailed analysis of one of these, alt1-1 (Al tolerant), suggests that this mutation positively impacts Al resistance in a manner dependent on pH adjustment rather than enhanced Al exclusion. Identification of these suppressor mutations, should not only further elucidate the biochemical and molecular mechanisms underlying Al toxicity and tolerance but also will develop a collection of mutations that may be useful for engineering crop plants that can grow and thrive in Al-toxic environments.

ATP-Binding Cassette Transporters↗

Mutational changes in physiochemical cell surface properties of plant-growth-stimulating Pseudomonas spp. do not influence the attachment properties of the cells.

Bacteriophage-resistant mutant strains of the root-colonizing Pseudomonas strains WCS358 and WCS374 lack the O-antigenic side chain of the lipopolysaccharide, as was shown by the loss of the typical lipopolysaccharide ladder pattern after analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These strains differed from their parent strains in cell surface hydrophobicity and in cell surface charge. The observed variation in these physicochemical characteristics could be explained by the differences in sugar composition. The mutant strains had no altered properties of adherence to sterile potato roots compared with their parental strains, nor were differences observed in the firm adhesion to hydrophilic, lipophilic, negatively charged, or positively charged artificial surfaces. These results show that neither physicochemical cell surface properties nor the presence of the O-antigenic side chain plays a major role in the firm adhesion of these bacterial cells to solid surfaces, including potato roots.

Antigens, Bacterial↗

Expression of a dominant negative mutant of epidermal growth factor receptor in the epidermis of transgenic mice elicits striking alterations in hair follicle development and skin structure.

Epidermal growth factor receptor (EGFR) is a key regulator of keratinocyte biology. However, the physiological role of EGFR in vivo has not been well established. To analyze the role of EGFR in skin, we have generated transgenic mice expressing an EGFR dominant negative mutant in the basal layer of epidermis and outer root sheath of hair follicles. Mice expressing the mutant receptor display short and waved pelage hair and curly whiskers during the first weeks of age, but subsequently pelage and vibrissa hairs become progressively sparser and atrophic. Eventually, most mice present severe alopecia. Histological examination of the skin of transgenic mice shows striking alterations in the development of hair follicles, which fail to enter into catagen stage. These alterations eventually lead to necrosis and disappearance of the follicles, accompanied by strong infiltration of the skin with inflammatory elements. The interfollicular epidermis of these mice shows marked hyperplasia, expression of hyperproliferation-associated keratin K6 and increased 5-bromo-2-deoxyuridine incorporation. EGFR function was inhibited in transgenic skin keratinocytes, since in vivo and in vitro autophosphorylation of EGFR was almost completely abolished on EGF stimulation. These results implicate EGFR in the control of hair cycle progression, and provide new information about its role in epidermal growth and differentiation.

Animals↗

Rop GTPase: a master switch of cell polarity development in plants.

Cell polarity is fundamentally important to plant growth and development, yet the mechanism governing its development is understood poorly. Several studies have revealed a role for Rop GTPases in pollen polar tip growth. Rop is also localized to the future site of root hair development and the tip of root hairs, and expression of constitutively active Rop mutants impacts on the morphogenesis of tip-growing root hairs as well as on non-tip-growing cells. These findings highlight the importance of Rop as a common switch in cell polarity control in plants.

Cell Polarity↗

Novel hydrotropism mutants of Arabidopsis thaliana and their altered waving response and phototropism.

Roots display positive hydrotropism in response to a moisture gradient, which is important for plants to escape from water stress and regulate the directional growth by interacting with other growth movements such as gravitropism, phototropism and waving response. On Earth, hydrotropism is interfered by gravitropism in particular, so that microgravity conditions or agravitropic mutants have been used for the study of hydrotropism. However, we have recently established an experimental system for the study of hydrotropism in Arabidopsis roots that easily develop hydrotropism in response to moisture gradient by overcoming gravitropism. Using the Arabidopsis system, we isolated hydrotropism mutants named root hydrotropism (rhy). In the present study, we examined the hydrotropism, gravitropism, phototropism, waving response and elongation growth of rhy4 and rhy5 roots that were defective in positive hydrotropism. Interestingly, rhy4 roots curved away from the water source and showed a reduced waving response. Both rhy4 and rhy5 showed normal gravitropism and a slight reduction in phototropism. These results suggest that there is a mutual molecular mechanism underlying hydrotropism, waving response and/or phototropism. Thus, we have obtained novel hydrotropic mutants that will be used for revealing molecular mechanism of root hydrotropism and its interaction with waving response and/or phototropism.

Arabidopsis↗

A role for the AKT1 potassium channel in plant nutrition.

In plants, potassium serves an essential role as an osmoticum and charge carrier. Its uptake by roots occurs by poorly defined mechanisms. To determine the role of potassium channels in planta, we performed a reverse genetic screen and identified an Arabidopsis thaliana mutant in which the AKT1 channel gene was disrupted. Roots of this mutant lacked inward-rectifying potassium channels and displayed reduced potassium (rubidium-86) uptake. Compared with wild type, mutant plants grew poorly on media with a potassium concentration of 100 micromolar or less. These results and membrane potential measurements suggest that the AKT1 channel mediates potassium uptake from solutions that contain as little as 10 micromolar potassium.

Arabidopsis↗

Arsenic uptake, translocation and speciation in pho1 and pho2 mutants of Arabidopsis thaliana.

Arsenate [As (V)] is taken up by phosphate [P (V)] transporters in the plasma membrane of roots cells, but the translocation of As from roots to shoots is not well understood. Two mutants of Arabidopsis thaliana (L.) [(pho1, P deficient) and (pho2, P accumulator)], with defects in the regulation and translocation of P (V) from roots to shoots, were therefore used in this study to investigate uptake, translocation and speciation of As in roots and shoots of plants grown in soil or nutrient solution. The shoots of the pho2 mutant contained higher P concentrations, but similar or slightly higher As concentrations, in comparison with the wild type. In the pho1 mutant, the P concentration in the shoots was lower, and the As concentration was higher, in comparison with the wild type. Both pho2 and the wild type contained mainly As (III) in roots and shoot (67-90% of total As). Arsenic was likely to be translocated by a different pathway to P (V) in the pho2 and pho1 mutants. Therefore, it is suggested that As (III) is the main As species translocated from roots to shoots in Arabidopsis thaliana.

Journal Article↗

Fractionation of carbohydrates in Arabidopsis root cell walls shows that three radial swelling loci are specifically involved in cellulose production.

Three non-allelic radial swelling mutants (rsw1, rsw2 and rsw3) of Arabidopsis thaliana L. Heynh. were shown to be specifically impaired in cellulose production. Fractionation methods that identify, characterise and quantify some of the major cell wall polysaccharides in small quantities of seedlings demonstrated that changes in the production of cellulose are much more pronounced than changes in the production of non-cellulosic polysaccharides. A crude cell wall pellet was sequentially extracted with chloroform methanol (to recover lipids), dimethyl sulphoxide (starch), ammonium oxalate (pectins) and alkali (hemicelluloses). Crystalline cellulose remained insoluble through subsequent treatments with an acetic/nitric acid mixture and with trifluoroacetic acid. Cetyltrimethylammonium bromide precipitation resolved neutral and acidic polymers in the fractions, and precipitation behaviour, monosaccharide composition and glycosidic linkage patterns identified the major polysaccharides. The deduced composition of the walls of wild-type seedlings and the structure and solubility properties of the major polymers were broadly typical of other dicots. The three temperature-sensitive, radial swelling mutants produced less cellulose in their roots than the wild type when grown at their restrictive temperature (31 degrees C). There were no significant differences at 21 degrees C where no radial swelling occurs. The limited changes seen in the monosaccharide compositions, glycosidic linkage patterns and quantities of non-cellulosic polysaccharides support the view that the RSW1, RSW2 and RSW3 genes are specifically involved in cellulose synthesis. Reduced deposition of cellulose was accompanied by increased accumulation of starch.

Arabidopsis↗

Galactose biosynthesis in Arabidopsis: genetic evidence for substrate channeling from UDP-D-galactose into cell wall polymers.

The biosynthesis of plant cell wall polysaccharides requires the concerted action of nucleotide sugar interconversion enzymes, nucleotide sugar transporters, and glycosyl transferases. How cell wall synthesis in planta is regulated, however, remains unclear. The root epidermal bulger 1 (reb1) mutant in Arabidopsis thaliana is partially deficient in cell wall arabinogalactan-protein (AGP), indicating a role for REB1 in AGP biosynthesis. We show that REB1 is allelic to ROOT HAIR DEFICIENT 1 (RHD1), one of five ubiquitously expressed genes that encode isoforms of UDP-D-glucose 4-epimerase (UGE), an enzyme that acts in the formation of UDP-D-galactose (UDP-D-Gal). The RHD1 isoform is specifically required for the galactosylation of xyloglucan (XG) and type II arabinogalactan (AGII) but is not involved either in D-galactose detoxification or in galactolipid biosynthesis. Epidermal cell walls in the root expansion zone lack arabinosylated (1-->6)-beta-D-galactan and galactosylated XG. In cortical cells of rhd1, galactosylated XG is absent, but an arabinosylated (1-->6)-beta-D-galactan is present. We conclude that the flux of galactose from UDP-D-Gal into different downstream products is compartmentalized at the level of cytosolic UGE isoforms. This suggests that substrate channeling plays a role in the regulation of plant cell wall biosynthesis.

Arabidopsis↗

Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons.

Herpes simplex virus type 1 (HSV-1) has many attractive features that suggest its utility for gene transfer to neurons. However, viral cytotoxicity and transient transgene expression limit practical applications even in the absence of viral replication. Mutant viruses deleted for the immediate early (IE) gene, ICP4, an essential transcriptional transactivator, are toxic to many cell types in culture in which only the remaining IE genes are expressed. In order to test directly the toxicity of other IE gene products in neurons and develop a mutant background capable of longterm transgene expression, we generated mutants deleted for multiple IE genes in various combinations and tested their relative cytotoxicity in 9L rat gliosarcoma cells, Vero monkey kidney cells, and primary rat cortical and dorsal root neurons in culture. Viral mutants deleted simultaneously for the IE genes encoding ICP4, ICP22 and ICP27 showed substantially reduced cytotoxicity compared with viruses deleted for ICP4 alone or ICP4 in combination with either ICP22, ICP27 or ICP47. Infection of neurons in culture with these triple IE deletion mutants substantially enhanced cell survival and permitted transgene expression for over 21 days. Such mutants may prove useful for efficient gene transfer and extended transgene expression in neurons in vitro and in vivo.

Animals↗