Mutant Shoots Research Articles

Disruption of the nitrate transporter genes AtNRT2.1 and AtNRT2.2 restricts growth at low external nitrate concentration.

The high-affinity transport systems in Arabidopsis thaliana (L.) Heynh. involve potentially seven genes. Among these, the AtNRT2.1 and/or AtNRT2.2 genes have been shown to play a major role in the inducible component of this transport system. The physiological impact of a disruption of AtNRT2.1 and AtNRT2.2 on plant growth and N-metabolism was investigated. The reduced nitrate uptake in the mutant under a limiting N-regime was found to correlate with a significant difference in shoot/root ratio between wild type and mutant and a drastically reduced nitrate level in the shoot of the mutant. Carbohydrate analyses of plants under a low nitrate supply revealed a slight increase in glucose and fructose in the mutant shoots as well as an increase in sucrose and starch contents in mutant shoots. Interestingly, the AtNRT2.4 and AtNRT2.5 genes were over-expressed in the mutant growing in reduced N-conditions, without any compensation by root nitrate influx. These results are discussed in the context of the putative role of the different NRT2 genes.

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.

Micrografting techniques for testing long-distance signalling in Arabidopsis.

Grafting in species other than Arabidopsis has generated persuasive evidence for long-distance signals involved in many plant processes, including regulation of flowering time and shoot branching. Hitherto, such approaches in Arabidopsis have been hampered by the lack of suitable grafting techniques. Here, a range of micrografting methods for young Arabidopsis seedlings are described. The simplest configuration was a single-hypocotyl graft, constructed with or without a supporting collar, allowing tests of root-shoot communication. More complex two-shoot grafts were also constructed, enabling tests of shoot-shoot communication. Integrity of grafts and absence of adventitious roots on scions were assessed using plants constitutively expressing a GUS gene as one graft partner. Using the max1 (more axillary growth) and max3 increased branching mutants, it was shown that a wild-type (WT) rootstock was able to inhibit rosette branching of mutant shoots. In two-shoot grafts with max1 and WT shoots on a max1 rootstock, the mutant shoot branched profusely, but the WT one did not. In two-shoot grafts with max1 and WT shoots on a WT rootstock, neither shoot exhibited increased branching. The results mirror those previously demonstrated in equivalent grafting experiments with the ramosus mutants in pea, and are consistent with the concept that a branching signal is capable of moving from root to shoot, but not from shoot to shoot. These grafting procedures will be valuable for revealing genes associated with many other long-distance signalling pathways, including flowering, systemic resistance and abiotic stress responses.

SEMAPHORE1 functions during the regulation of ancestrally duplicated knox genes and polar auxin transport in maize.

The expression of class 1 knotted1-like homeobox (knox) genes affects numerous plant developmental processes, including cell-fate acquisition, lateral organ initiation, and maintenance of shoot apical meristems. The SEMAPHORE1 gene product is required for the negative regulation of a subset of maize knox genes, the duplicated loci rough sheath 1 and gnarley1 (knox4). Recessive mutations in semaphore1 result in the ectopic expression of knox genes in leaf and endosperm tissue. Genetic analyses suggest that SEMAPHORE1 may regulate knox gene expression in a different developmental pathway than ROUGH SHEATH2, the first-identified regulator of knox gene expression in maize. Mutations at semaphore1 are pleiotropic, disrupting specific domains of the shoot. However, unlike previously described mutations that cause ectopic knox gene expression, semaphore1 mutations affect development of the embryo, endosperm, lateral roots, and pollen. Moreover, polar transport of the phytohormone auxin is significantly reduced in semaphore1 mutant shoots. The data suggest that many of the pleiotropic semaphore1 phenotypes result from defective polar auxin transport (PAT) in sem1 mutant shoots, and support models correlating down-regulated knox gene expression and PAT in maize shoots.

Cuttings of the non-rooting rac tobacco mutant overaccumulate phenolic compounds.

The auxin and phenolic contents, as well as phenylalanine ammonia-lyase (PAL) activity, were determined in in vitro cultured shoots of the recalcitrant-to-root rac mutant of tobacco, and compared with wild-type shoots. The mutant and wild-type shoots showed similar auxin changes during the culture cycle, but with higher contents for the mutant. A transient peak of auxin (corresponding to the achievement of the rooting inductive phase) occurred at day 14 in both types of shoots, but earlier in the basal parts of the wild-type stems. The rac shoots contained more phenolics, corresponding with an increased PAL activity. The most abundant phenolic compound found in the two types of tobacco was chlorogenic acid, which was more abundant in the rac shoots. Rutin was also detected at a higher concentration in the mutant shoots. Basal parts of wild-type shoots treated with 10-3 M chlorogenic acid reacted by accumulating auxins and, unlike untreated controls, did not form adventitious roots. The relationships between these biochemical analyses in relation to the growth limitation of the rac mutant, and the inhibition of its root development, are discussed.

NAA Restores Apical Dominance in the axr3-1 Mutant of Arabidopsis thaliana

Strong evidence for a role of auxin in apical dominance is provided by the classic Thimann-Skoog experiment (Proceedings of the National Academy of Sciences, USA19: 714–716, 1933) wherein exogenous auxin applied to a decapitated shoot represses outgrowth of the next lower lateral bud. Although apical dominance in most herbaceous species can be restored by this auxin treatment, such is not the case with wild-type Arabidopsis thaliana. In the present study, it has been demonstrated that apical dominance can be partially or fully restored with exogenous auxin (1% naphthaleneacetic acid, NAA) applied to the decapitated shoot of the axr3-1 mutant which is thought to be hypersensitive to auxin. A similar repressive response to auxin (1μM NAA) was also shown in an in vitro assay with detached nodes. The role of AXR3 as a gene mediating auxin response is thus supported.

Auxin inhibition of decapitation-induced branching is dependent on graft-transmissible signals regulated by genes Rms1 and Rms2.

Decapitation-induced axillary bud outgrowth is a vital mechanism whereby shoots are able to continue normal growth and development. In many plants, including wild-type garden pea (Pisum sativum L.), this process can be inhibited by exogenous auxin. Using the ramosus (rms) increased branching mutants of pea, we present evidence that this response to auxin is dependent on graft-transmissible substance(s) regulated by the genes Rms1 and Rms2. The response to exogenous auxin is massively diminished in decapitated rms1 and rms2 mutant plants. However, basipetal auxin transport is not reduced in intact or decapitated mutants. Grafting rms1 or rms2 shoots onto wild-type rootstocks restored the auxin response, indicating that Rms1 and Rms2 gene action in the rootstock is sufficient to enable an auxin response in mutant shoots. We conclude that Rms1 and Rms2 act in the rootstock and shoot to control levels of mobile substance(s) that interact with exogenous auxin in the inhibition of bud outgrowth after decapitation. At least for rms1, the reduced auxin response is unlikely to be due to an inability of auxin to decrease xylem sap cytokinin content, as this is already low in intact rms1 plants. Consequently, we have genetic evidence that auxin action in decapitated plants depends on at least one novel long-distance signal.

The recalcitrance to rooting of the micropropagated shoots of the rac tobacco mutant: Implications of polyamines and of the polyamine metabolism

Rooting of wild-type tobacco ( Nicotiana tabacum cv. Xanthi) shoots raised in vitro was promoted by polyamines in the absence of any other growth regulator and was inhibited by two inhibitors of polyamine metabolism. The auxin insensitive and recalcitrant to rooting rac mutant shoots did not respond to the same treatments. The activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxidase (DAO), polyamine oxidase (PAO) and transglutaminases (TGases), and the titres of free and conjugated polyamines were estimated in the whole shoots and the basal parts of the stems of both tobaccos in the course of multiplication in vitro. The rac shoots grew at a lower rate. The wild-type rooted from the 7th day without special treatment. During the second week of culture, the shoots of both tobaccos were actively growing and showed an increase in ADC, ODC, DAO, PAO and TGase activities. Afterwards all these activities declined. These changes were concomitant with an increase in the polyamine contents (free and conjugated). Biosynthesis and oxidation of polyamines apparently occurred simultaneously and seemed directly correlated. In the basal part of the mutant stems however, the accumulation of free and conjugated putrescine as well as the transient increase in biosynthetic enzyme activities were delayed compared to the wild-type. These results are discussed in relation to growth behaviour and to root formation.

IAA-oxidase activity and auxin protectors in nonrooting, rac, mutant shoots of tobacco in vitro

The peroxidase and IAA-oxidase activities, the degree of auxin protection and the amount of soluble phenolics were determined in in vitro cultured shoots of a nonrooting mutant, rac, of tobacco compared to its wild homologue. The mutant and wild shoots showed similar peroxidase variations along the growth cycle of 21 days, but with higher levels of activity for the rac mutant. During this growth cycle, the minimum of peroxidase activity occurred at day 14 for both tobacco whole shoots. However, this minimum of activity did not occur at the same day in the basal part of the stem, where roots may appear, of the two types of tobacco. Both mutant and wild whole shoots showed about the same IAA-oxidase activity in the fractions resulting from a gel filtration of the crude extracts through a Sephadex G-100 column but differed in the degree of auxin protection. The rac shoots exhibited a very high level of auxin protectors of low molecular weight, among which chlorogenic acid. They were also characterized by eight to nine times higher level of soluble phenolics. The relationships between these biochemical aspects in relation to the absence of root formation in the rac mutant are discussed.

Synthesis of Some C28 Steroids with C-24 (28) Double Bonds

Authentic samples for identification of steroids in the shoots of dwarf mutant of Arabidopsis thaliana L., 24-methylcholesta-4, 24 (28) -dien-3-one and 24-methyl-5α-cholest-24 (28) -en-3β-ol were synthesized using olefination with Tebbe reagent as a key reaction. Related steroids, 24-methylcholesta-4, 24 (28) -dien-3β-ol and 24-methyl-5α-cholest-24 (28) -en-3-one, were prepared.

The response of lazy-2 tomato seedlings to curvature-inducing magnetic gradients is modulated by light.

Shoots of the lazy-2 mutant of tomato (Lycopersicon esculentum Mill., cv. Ailsa Craig) exhibit negative gravitropism in the dark, but respond positively gravitropically in (red) light. In order to test whether high-gradient magnetic fields (HGMFs) exert only ponderomotive effects on amyloplasts or affect other physiological processes, we induced magnetophoretic curvature in wild-type (WT) and lazy-2 mutant seedlings. Straight hypocotyls of 4-d-old plants were selected and the tips of their hooks were placed in an HGMF near the edge of a magnetized ferromagnetic wedge [grad (H2/2) approximately 10(9)-10(10) Oe2/cm] and mounted on a 1-rpm clinostat. After 4 h in the dark, 85% of WT hypocotyls and 67% of mutant hypocotyls curved toward the wedge. When the seedlings were exposed to red light for 1 h prior to and during the application of the HGMF, 78% of the WT seedlings curved toward the magnetic gradient, but the majority of the lazy-2 seedlings (75%) curved away from the stronger field area. Intracellular amyloplast displacement in the HGMF was similar for both varieties and resembled the displacement after horizontal reorientation. The WT showed a distinct graviresponse pattern depending on the orientation of the hook, even after excision of the apex. Application of HGMFs to decapitated hypocotyls resulted in curvature consistent with that obtained after horizontal reorientation. After light exposure, decapitated lazy-2 seedlings did not respond positively gravitropically. The data imply that the lazy-2 mutants perceive the displacement of amyloplasts in a similar manner to the WT and that the HGMF does not affect the graviresponse mechanism. The study demonstrates that ponderomotive forces due to HGMFs are useful for the analysis of the gravity-sensing mechanism in plants.

Evaluation of sole nitrogen sources for shoot and leaf disc cultures of sugarbeet

Use of single nitrogen sources in nutrient media is essential to ascertaining the relative role and regulation of nitrogen assimilatory steps, and may help identify and understand highly productive media for micropropagation and adventitious shoot formation. Eight endogenous nitrogen-containing ions or compounds in sugarbeet (nitrate, ammonium, glutamine, glutamate, urea, proline, glycine betaine and choline) were examined for ability to serve as sole nitrogen source for shoot or leaf disc culture of sugarbeet (Beta vulgaris L.) model clone REL-1. The most productive concentrations of nitrate, ammonium, urea, and glutamine as sole nitrogen sources were moderately supportive of shoot multiplication (64, 70, 81 and 71%, respectively) and fresh weight increase (65, 41, 54 and 41%, respectively) compared to shoot culture growth with the Murashige-Skoog nitrogen mix of 40 mM nitrate and 20 mM ammonium. Glutamate and proline were at best poorly supportive, and glycine betaine and choline were non-supportive. Callus initiation from leaf discs was supported only by nitrate, ammonium, urea, glutamine and proline (50, 100, 100, 100 and 80%, respectively, at the best concentrations, of that on Murashige-Skoog medium). Subsequent shoot regeneration from the intact disc callus in those cultures only occurred on media with nitrate, urea, glutamine, or proline (12, 3, 28 and 3% as many shoots, respectively, as on Murashige-Skoog medium). Overall, the Murashige-Skoog nitrogen mix was superior or equal to any single nitrogen source. However, single nitrogen source media with nitrate, ammonium, urea, glutamine or proline should have significant utility for shoot or leaf disc cultures of mutants with impaired nitrogen assimilation, in comparative physiology studies, or in dual cultures with pathogens of limited ability to use any of these forms of nitrogen.

Management of chimera through direct shoot regeneration from florets of chrysanthemum (Chrysanthemum morifolium Ramat.)

SummaryThe main bottleneck of mutation induction in vegetatively propagated ornamentals is the development of chimera. Ephemeral small sectors apparent on petals cannot generally be recovered as solid mutant shoots. An efficient technique has been standardized in the present experiment for direct shoot regeneration from individual florets of...chrysanthemum (Chrysanthemum morifolium Ramat.) cv. Colchi Bahar. The efficiency of direct shoot regeneration was tested on 16 media based on MS basal medium containing sucrose, agar and different concentrations and combinations of BAP and NAA. The regenerative potential varied with the developmental stages of the flower head. The highest percentage of shoot formation and most shoots per responding explant were observed on florets taken from stage II flower heads. Direct shoot formation was confirmed by scanning electron microscopy and histological observations. The technique has been tested on 16 other cultivars in the medium containing 0.2 mg l21 NAA and 1.mg.l1 BAP. Gamma ray of 1.5 and 2.0 krad induced yellow and white floret colour sectors in `Maghi' (original colour-mauve) have been established in pure form using this technique. The regenerated plants were rooted in vitro and successfully transferred to soil where they grew vigorously and flowered true to the floret colour type.

Peroxidase activity, ethylene production, lignification and growth limitation in shoots of a nonrooting mutant of tobacco

The rooting recalcitrant rac Nicotiana tabacum cv Xanthi mutant has been multiplied in vitro under the form of shoots in parallel to wild-type. rac Shoots grew at a lower rate and did not root whatever the treatments when compared to those of wild-type shoots. They were characterized by a higher lignin level, a higher total specific peroxidase activity with higher activity of both acidic and basic isoperoxidases (although missing and supernumerary isoenzymes were observed), and higher ethylene production. These observations might be causally related to growth inhibitions as similar incidences have been noted in different stress-induced growth limitation, through cell wall rigidification and auxin catabolism. The relationship between these aspects and rooting recalcitrance remains to be explored.

CLAVATA2, a regulator of meristem and organ development in Arabidopsis.

Mutations at the CLAVATA2 (CLV2) locus of Arabidopsis result in enlarged shoot and flower meristems, as well as alterations in the development of the gynoecia, flower pedicels, and stamens. The shoot and flower meristem phenotypes of clv2 mutants are similar to weak clv1 and clv3 mutants. We present genetic analysis that CLV2 may function in the same pathway as CLV1 and CLV3 in the regulation of meristem development, but function separately in the regulation of organ development. We also present evidence that clv2 phenotypes are altered when the mutants are grown under short-day light conditions. These alterations include flower-to-shoot transformations, as well as a nearly complete suppression of the flower phenotypes, indicating that the requirement for CLV2 changes in response to different physiological conditions. The stm-1 mutation dominantly suppresses clv2, and clv2 mutations suppress the strong stm-1 allele, but not the weak stm-2 allele.

Regulation of leaf initiation by the terminal ear 1 gene of maize.

Higher plants elaborate much of their architecture post-embryonically through development initiated at the tips of shoots. During vegetative growth, leaf primordia arise at predictable sites to give characteristic leaf arrangements, or phyllotaxies. How these sites are determined is a long-standing question that bears on the nature of pattern-formation mechanisms in plants. Fate-mapping studies in several species indicate that each leaf primordium becomes organized from a group of 100-200 cells on the flank of the shoot apex. Although molecular studies indicate that the regulated expression of specific homeobox genes plays some part in this determination process, mechanisms that regulate the timing and position of leaf initiation are less well understood. Here we describe a gene from maize, terminal ear 1. Patterns of expression of this gene in the shoot and phenotypes of mutants indicate a role for terminal ear 1 in regulating leaf initiation. The tel gene product contains conserved RNA-binding motifs, indicating that it may function through an RNA-binding activity.

Blockage of Brassinosteroid Biosynthesis and Sensitivity Causes Dwarfism in Garden Pea.

Endogenous brassinosteroids (BRs) in the dwarf mutants lka and lkb of garden pea (Pisum sativum L.) and comparable wild-type plants were quantified by gas chromatography-selected ion monitoring using deuterated internal standards. In young shoots of the lkb mutant, the levels of brassinolide, castasterone, and 6-deoxocastasterone were 23-, 22-, and 9-fold lower, respectively, than those of wild-type plants. Applications of brassinolide, castasterone, typhasterol, 3-dehydroteasterone, and teasterone normalized internode growth of lkb seedlings. These findings indicate that the lkb plants are BR-deficient mutants, probably as a consequence of a block in the BR biosynthetic pathway prior to the production of teasterone. Young shoots of lka plants contained only 50% less brassinolide and 5 times more castasterone than the equivalent wild-type tissues. The lka seedlings were approximately 100 times less responsive to brassinolide than the lkb mutant, and application of castasterone had only a marginal effect on lka internode growth, suggesting that the lka lesion results in impaired sensitivity to BR.

Phenotype, Inheritance, and Regulation of Expression of a New Virescent Mutant in Watermelon: Juvenile Albino

Juvenile albino, gene symbol ja, is a spontaneous virescent mutant, first observed in `Dixielee' and an F2 population of `G17AB' (msms) × `Dixielee' in 1992. Hypocotyls, new young leaves, shoot tips, tendrils and flowers on the main shoot of the ja mutant are all albino during early spring. The interior portions of albino leaves gradually become green, while the margins remain albino. Fruit rind color of the mutant is variegated. Growth of the ja mutant is severely impaired in the early spring. However, the mutant grows at a rate comparable to wild-type in the summer, and produces fruit of almost normal size. Genetic analysis of F1, F2, and BC1 populations derived from the ja mutant showed that the gene for the ja mutant is inherited as a single, recessive, nuclear gene. Segregation ratios in the F2 and BC1 progenies derived from the cross between the previously reported delayed green virescent mutant and the ja mutant indicate independent inheritance of the genes dg and ja. Temperature and red/far-red light had no differential effect on mutant and the wild-type plants. An increase of daylength from 8 to 15 hours increased fresh weight and chlorophyll content more in the ja mutant than in the wild-type. The mutant had a higher chlorophyll a: b ratio than the wild-type under long days. Chlorophyll synthesis or accumulation in the mutant is severely impaired under short days. This is the only virescent mutant in the family Cucurbitaceae whose expression is regulated by daylength.

Differential gene expression in salt-tolerant rice mutant and its parental variety

The differential expressions of three genes rbcL, salT and rab!6 in response to ABA, NaCl, PEG and heat shock were investigated in seedlings of a salt-tolerant rice mutant 20 (mutant 20) and its parental variety Oryza sativa var. japonica 77-170(170). By Northern blot analysis it was found that ABA induced the expression of all three genes of rbcL, salT and rab16 in shoots and roots of both 170 and mutant 20 with the exceptions of rab16 in shoots of mutant 20 and rbcL in roots of 170. Lower concentrations of NaCl induced rbcL expression in shoots of mutant 20 but not 170. Higher concentrations of NaCl decreased rbcL expression but induced expressions of salT and rab16 in shoots of both 170 and mutant 20. PEG(15%) and 37℃ heat shock showed almost no effects on the expression of the three genes in mutant 20. However, they caused a decrease in rbcL expression and slight induction of the rab16 gene in 170, with salT expression unaffected. These results indicated that mutant 20 was relatively less responsive to applied hormonal and environmental factors as compared with 170, suggesting that mutant 20 might have acquired mechanisms by which the plant is less responsive to environmental stresses and hence gain a stronger ability to tolerate stresses.

Juvenile albino, a Virescent Mutant in Watermelon: Inheritance and Expression

juvenile albino (ja) is a spontaneous mutant, first observed in 1992. Hypocotyls, new young leaves, shoot tips, tendrils, and flowers on the main shoot of the ja mutant are all albino during early spring and late fall. The interior of the albino leaves gradually become green, while the margins remain albino. Fruit rind color of the mutant is variegated. Growth of the ja mutant is severely impaired in the early spring and late fall. However, the mutant grows almost normal in the summer, and produces fruits of almost normal size. Genetic analysis of F1, F2, and BC1 populations derived from the ja mutant showed that ja mutant is inherited as a single, recessive, nuclear gene. The segregation ratios in the F2 and BC1 progenies derived from the cross between the previously reported dg virescent mutant and the ja mutant indicated that both are inherited independently. Experiments with temperature (3–5C vs. 20–22C at night), day length (8 vs. 15 h), and red and/or far-red light (15 vs. 0 min) at the end of an 8-h day were performed to investigate the regulation of ja trait expression. Temperature and red/far-red light had no differential effect on mutant and wild-type plants. However, significantly increased fresh weight and chlorophyll content were observed in the ja mutant over the wild-type when grown under long-day conditions. In addition, chlorophyll synthesis or accumulation in the mutant is severely impaired under short-day conditions. To our knowledge, this is the only virescent mutant in Cucurbitaceae whose expression is regulated by day length.