Reduced Cell Elongation Research Articles

Identification of a protein, YneA, responsible for cell division suppression during the SOS response in Bacillus subtilis.

A knock-out mutant of the dinR gene that encodes the SOS regulon repressor in Bacillus subtilis was constructed. The yneA, yneB and ynzC genes transcribed divergently from the dinR gene were strongly induced in mutant cells. Northern hybridization analyses revealed that these genes collectively form an operon and belong to the SOS regulon. The simultaneous deletion of dinR and yneA suppressed the filamentous phenotype of the dinR mutant. Furthermore, although yneA is suppressed in the wild-type cell in the absence of SOS induction, artificial expression of the YneA protein using an IPTG-inducible promoter resulted in cell elongation. Disruption of yneA significantly reduced cell elongation after the induction of the SOS response by mitomycin C in dinR+ cells. These results indicate that the YneA protein is responsible for cell division suppression during the SOS response in B. subtilis. Localization of the FtsZ protein to the cell division site was reduced in dinR-disrupted or yneA-expressing cells, further suggesting that the YneA protein suppresses cell division through the suppression of FtsZ ring formation. Interestingly, the B. subtilis YneA protein is structurally and phylogenetically unrelated to its functional counterpart in Escherichia coli, SulA.

Alteration of oriented deposition of cellulose microfibrils by mutation of a katanin-like microtubule-severing protein.

It has long been hypothesized that cortical microtubules (MTs) control the orientation of cellulose microfibril deposition, but no mutants with alterations of MT orientation have been shown to affect this process. We have shown previously that in Arabidopsis, the fra2 mutation causes aberrant cortical MT orientation and reduced cell elongation, and the gene responsible for the fra2 mutation encodes a katanin-like protein. In this study, using field emission scanning electron microscopy, we found that the fra2 mutation altered the normal orientation of cellulose microfibrils in walls of expanding cells. Although cellulose microfibrils in walls of wild-type cells were oriented transversely along the elongation axis, cellulose microfibrils in walls of fra2 cells often formed bands and ran in different directions. The fra2 mutation also caused aberrant deposition of cellulose microfibrils in secondary walls of fiber cells. The aberrant orientation of cellulose microfibrils was shown to be correlated with disorganized cortical MTs in several cell types examined. In addition, the thickness of both primary and secondary cell walls was reduced significantly in the fra2 mutant. These results indicate that the katanin-like protein is essential for oriented cellulose microfibril deposition and normal cell wall biosynthesis. We further demonstrated that the Arabidopsis katanin-like protein possessed MT-severing activity in vitro; thus, it is an ortholog of animal katanin. We propose that the aberrant MT orientation caused by the mutation of katanin results in the distorted deposition of cellulose microfibrils, which in turn leads to a defect in cell elongation. These findings strongly support the hypothesis that cortical MTs regulate the oriented deposition of cellulose microfibrils that determines the direction of cell elongation.

KOBITO1 encodes a novel plasma membrane protein necessary for normal synthesis of cellulose during cell expansion in Arabidopsis.

The cell wall is the major limiting factor for plant growth. Wall extension is thought to result from the loosening of its structure. However, it is not known how this is coordinated with wall synthesis. We have identified two novel allelic cellulose-deficient dwarf mutants, kobito1-1 and kobito1-2 (kob1-1 and kob1-2). The cellulose deficiency was confirmed by the direct observation of microfibrils in most recent wall layers of elongating root cells. In contrast to the wild type, which showed transversely oriented parallel microfibrils, kob1 microfibrils were randomized and occluded by a layer of pectic material. No such changes were observed in another dwarf mutant, pom1, suggesting that the cellulose defect in kob1 is not an indirect result of the reduced cell elongation. Interestingly, in the meristematic zone of kob1 roots, microfibrils appeared unaltered compared with the wild type, suggesting a role for KOB1 preferentially in rapidly elongating cells. KOB1 was cloned and encodes a novel, highly conserved, plant-specific protein that is plasma membrane bound, as shown with a green fluorescent protein-KOB1 fusion protein. KOB1 mRNA was present in all organs investigated, and its overexpression did not cause visible phenotypic changes. KOB1 may be part of the cellulose synthesis machinery in elongating cells, or it may play a role in the coordination between cell elongation and cellulose synthesis.

The role of gibberellins in embryo axis development.

The role of gibberellins (GAs) during early embryo development was examined using microspore-derived embryos (MDEs) of Brassica napus. At the globular stage of development, 10 d after initial culture (DAC) when endogenous GA(1) levels are increasing rapidly, a triazole, uniconazole, was used at 1, 33 and 100 microM to inhibit GA biosynthesis. Within this dose range there was no apparent effect of the inhibitor on embryo growth through to the early torpedo stage. However, by 25 DAC uniconazole-treated MDEs showed significantly reduced (50%) axis elongation. Addition of GA(1) at 33 microM on 14 DAC to embryos pretreated with 1 microM uniconazole on 10 DAC prevented this reduction in axis length, giving axis elongation equivalent to untreated MDEs. Application of GA(1) alone, however, did not significantly increase axis elongation. The reduced axis growth seen with uniconazole treatment was due to reduced cell elongation, but not cell number, and the co-applied GA(1) thus prevented the uniconazole-induced reduction in cell length. The elongating axis of MDEs may thus be a useful tool for examining the role of GAs in cell elongation.

Lilliputian Mutant of Maize Lacks Cell Elongation and Shows Defects in Organization of Actin Cytoskeleton

The maize mutant lilliputian is characterized by miniature seedling stature, reduced cell elongation, and aberrant root anatomy. Here, we document that root cells of this mutant show several defects in the organization of actin filaments (AFs). Specifically, cells within the meristem lack dense perinuclear AF baskets and fail to redistribute AFs during mitosis. In contrast, mitotic cells of wild-type roots accumulate AFs at plasma membrane-associated domains that face the mitotic spindle poles. Both mitotic and early postmitotic mutant cells fail to assemble transverse arrays of cortical AFs, which are characteristic for wild-type root cells. In addition, early postmitotic cells show aberrant distribution of endoplasmic AF bundles that are normally organized through anchorage sites at cross-walls and nuclear surfaces. In wild-type root apices, these latter AF bundles are organized in the form of symmetrically arranged conical arrays and appear to be essential for the onset of rapid cell elongation. Exposure of wild-type and cv. Alarik maize root apices to the F-actin drugs cytochalasin D and latrunculin B mimics the phenotype of lilliputian root apices. In contrast to AFs, microtubules are more or less normally organized in root cells of lilliputian mutant. Collectively, these data suggest that the LILLIPUTIAN protein, the nature of which is still unknown, impinges on plant development via its action on the actin cytoskeleton.

Phosphate availability regulates root system architecture in Arabidopsis.

Plant root systems are highly plastic in their development and can adapt their architecture in response to the prevailing environmental conditions. One important parameter is the availability of phosphate, which is highly immobile in soil such that the arrangement of roots within the soil will profoundly affect the ability of the plant to acquire this essential nutrient. Consistent with this, the availability of phosphate was found to have a marked effect on the root system architecture of Arabidopsis. Low phosphate availability favored lateral root growth over primary root growth, through increased lateral root density and length, and reduced primary root growth mediated by reduced cell elongation. The ability of the root system to respond to phosphate availability was found to be independent of sucrose supply and auxin signaling. In contrast, shoot phosphate status was found to influence the root system architecture response to phosphate availability.

The Arabidopsis compact inflorescence genes: phase-specific growth regulation and the determination of inflorescence architecture.

During their life cycle, higher plants pass through a series of growth phases that are characterized by the production of morphologically distinct vegetative and reproductive organs and by different growth patterns. Three major phases have been described in Arabidopsis: juvenile vegetative, adult vegetative, and reproductive. In this report we describe a novel, phase-specific mutant in Arabidopsis, compact inflorescence (cif). The most apparent aspect of the cif phenotype is a strong reduction in the elongation of internodes in the inflorescence, resulting in the formation of a floral cluster at the apical end of all reproductive shoots. Elongation and expansion of adult vegetative rosette leaves are also compromised in mutant plants. The onset of the cif trait correlates closely with morphological changes marking the phase transition from juvenile to adult, and mutant plants produce normal flowers and are fully fertile. Hence the cif phenotype appears to be adult vegetative phase-specific. Histological sections of mutant inflorescence internodes indicate normal tissue specification, but reduced cell elongation compared to wild-type. compact inflorescence is inherited as a two-gene trait involving the action of a recessive and a dominant locus. These two cif genes appear to be key components of a growth regulatory pathway that is closely linked to phase change, and specifies critical aspects of plant growth and architecture including inflorescence internode length.

Effect of Gibberellic Acid on Cell Division and Cell Elongation in Anthers of the Gibberellin Deficient gib‐1 Mutant of Tomato

Abstract: Analysis of flower development in the gibberellin (GA) deficient gib‐1 tomato mutant showed that while flower development is arrested the anther growth slowed down unless GA was applied. Northern blot analyses showed that treatment with GA3 enhanced the expression of genes which are correlated with DNA replication (histones h1 and h2b) and cell elongation (expansin and α‐tubulin). In anthers, at developmental arrest, cell elongation was affected in the tapetum, pollen mother cells and epidermal cells. Elongation was significantly less in the longitudinal direction, while in the transverse direction outer tapetum and pollen mother cells were also smaller. Except for the outer tapetum, the cell numbers were the same as in wild‐type tomato, indicating that cell division was less influenced. Scanning electron microscopy revealed that on the lateral surface of gib‐1 anthers the epidermal cells that were affected in their elongation due to GA deficiency did not differentiate into interlocking hairs. Induction of hair development, of which the timing was similar to wild type, occurred after treatment with GA3, GA1, GA4 and GA9, while GA20 was less effective. Analysis of the in situ localization of expansin and histone h2b transcripts in gib‐1 anthers after GA3 treatment revealed that the increased mRNA accumulation was predominantly localized in the cells that were less elongated compared to wild‐type cells. The consequence of reduced cell elongation due to GA deficiency on anther development is discussed.

Quantitative trait loci on chromosome 4B for coleoptile length and early vigour in wheat (Triticum aestivum L.)

The Norin-10 dwarfing genes, Rht-B1b (Rht1) and Rht-D1b (Rht2), are commonly used to reduce plant height and increase grain yield in wheat breeding programs. These dwarfing genes lower sensitivity of vegetative tissue to endogenous gibberellin to reduce cell and subsequent stem elongation. This reduction in cell elongation capacity reportedly results in a concomitant reduction in coleoptile length and early vigour (leaf area) thereby affecting seedling establishment and growth. A detailed genetic map from a cross between tall Halberd (Rht-B1a) and semidwarf Cranbrook (Rht-B1b) wheat cultivars was used to assess genetic factors affecting seedling growth. Parental and 150 doubled haploid progeny lines were characterised for seedling and height-related traits in controlled and field environments. Genotypic variation was large and predominantly under additive genetic control with evidence for transgressive segregation for some traits. Narrow-sense heritabilities were moderate to high (h2 = 0.31–0.91) indicating a strong genetic basis for differences between progeny. Molecular marker analyses identified a number of significant (P < 0.05) quantitative trait loci (QTL) for each trait. A major QTL, mapping directly to the Rht-B1 locus on chromosome arm 4BS, accounted for up to 49% of the genotypic variance in peduncle length and plant height, and 27–45% of the genotypic variance in coleoptile length across different temperatures. Another QTL, located close to the RFLP marker XksuC2 on the long arm of chromosome 4B, accounted for 15–27% of the genotypic variance in coleoptile length. The influence of the XksuC2-linked QTL on coleoptile length was greatest at 19˚C and decreased with cooler temperatures. The same QTL affected reductions in leaf size, and both coleoptile tiller size and presence to affect overall seedling vigour. There was also some evidence for epistatic interactions influencing coleoptile tiller growth. Reductions in plant size at the Rht-B1b and XksuC2 loci were associated with presence of the Cranbrook 4B allele. The negative genetic effect of the Rht-B1b dwarfing gene on early growth of wheat confirms phenotypic evidence of a pleiotropic effect of Rht-B1b on establishment and early vigour. Genetic increases in coleoptile length and early leaf area development are likely to be limited in wheat populations containing the Rht-B1b dwarfing gene.

Gibberellin and ethylene control endoreduplication levels in the Arabidopsis thaliana hypocotyl.

We have previously shown that endoreduplication levels in hypocotyls of Arabidopsis thaliana (L.) Heynh. are under negative control of phytochromes. In this study, the hormonal regulation of this process was analysed using a collection of A. thaliana mutants. The results show that two hormones in particular, gibberellin (GA) and ethylene, play distinct roles. Hypocotyl cells of the GA-deficient mutant ga1-11 grown in the dark did not elongate and showed a greatly reduced endoreduplication. Normal endoreduplication could be restored by supplying 10(-9) M of the gibberellin GA(4+7), whereas the restoration of normal cell growth required 100-fold higher concentrations. The GA-insensitive mutant gai showed reduced cell elongation but normal ploidy levels. We conclude that (i) GA(4+7) has a global positive effect on endoreduplication and (ii) that endoreduplication is more sensitive to GA(4+7) than cell elongation. Ethylene had a completely different effect. It induced an extra round of endoreduplication both in light- and dark-grown seedlings and acted mainly on discrete steps rather than having a global effect on endoreduplication. The genes EIN2 and CTR1, components of the ethylene signal transduction pathway were both involved in this process.

A role for arabinogalactan-proteins in plant cell expansion: evidence from studies on the interaction of beta-glucosyl Yariv reagent with seedlings of Arabidopsis thaliana.

Seedlings of Arabidopsis thaliana were germinated and grown in medium containing beta-glucosyl Yariv reagent (beta GlcY), a synthetic phenyl glycoside that interacts specifically with arabinogalactan-proteins (AGPs), a class of plant cell surface proteoglycans. The effect of beta GlcY on the seedlings was to reduce the overall growth of both the root and the shoot. beta GlcY only accumulated in the root tissues and the reduced growth of the shoot appeared to be an indirect effect of impaired root growth. Reduced root growth was a consequence of a reduction in cell elongation during the postproliferation phase of elongation at the root apex and this was associated with extensive radial expansion of root epidermal cells. beta GlcY penetrated roots as far as the endodermis and it is suggested that the interaction of beta GlcY with AGPs in the load-bearing cell layers inhibited root elongation. When beta GlcY was added to carrot suspension-cultured cells that had been induced to elongate rather than proliferate, cell elongation was inhibited. The AGP-unreactive alpha-galactosyl Yariv reagent (alpha GalY) had no biological activity in either of these systems.

Role of the cytoskeleton in the reaction of fibroblasts to multiple grooved substrata

The role of the cytoskeleton and cell attachments in the alignment of baby hamster kidney fibroblasts to ridge and groove substratum topography was investigated using confocal scanning microscopy. This was carried out with normal cells and cells treated with the cytoskeleton modifiers cytochalasin D, colcemid, and taxol. Actin was localised with fluorescent phalloidin. Tubulin, vinculin, and intracellular adhesion molecule-1 were visualised by indirect immunofluorescence. The spreading, elongation, and orientation of the cells after 24 h of culture in these conditions were measured on grooves of 5, 10, and 25 microns width and 0.5, 1, 2, and 5 microns depth. We have also observed events over the first 30 min of cell attachment. Five minutes after cell attachment, F-actin condensations were seen close to the intersection of groove wall and ridge top, that is, at a topographic discontinuity. The condensations were often at right angles to the groove edge and showed a periodicity of 0.6 microns. Vinculin arrangement at the early stages of cell spreading was similar to that of actin. Organisation of the microtubule system followed later, becoming obvious at about 30 min after cell plating. The Curtis and Clark theory (that cells react to topography primarily at lines of discontinuity in the substratum by actin nucleation) is supported by these results. The use of cytoskeletal poisons did not entirely abolish cell reaction to grooves. Colcemid increased cell spreading and reduced cell orientation and elongation. Cytochalasin D reduced cell spreading, orientation, and elongation. Taxol reduced cell elongation but did not affect cell spreading and orientation. We conclude that the aggregation of actin along groove/ridge boundaries is a primary driving event in determining fibroblast orientation on microgrooved substrata.

Modification by Ethylene of the Cell Growth Pattern in Different Tissues of Etiolated Lupine Hypocotyls

The influence of ethylene on growth in etiolated lupine (Lupinus albus L.) hypocotyls was studied in ethephon-treated plants. Ethephon reduced the length and increased the diameter of hypocotyls. At the end of the hypocotyl growth period (14 days), the fresh weight was reduced by 53%, and the dry weight was reduced by 16%. Thus, ethylene reduced water uptake in the tissues to a greater extent than the incorporation of new materials. Light microscopic measurements showed that the thickness of tissues was stimulated by ethylene, the vascular cylinder and cortex exhibiting greater increases (55 and 45%, respectively) than pith (26%) or epidermis (12%). Ethephon modified the cell growth pattern, stimulating lateral cell expansion and cell wall thickness, while reducing cell elongation. The response to ethylene varied in the different tissues and was higher in cortex and pith cells than in the epidermis cells. The ethylene-induced cell expansion in the cortex varied according to the localization of cells in the tissue: the central and subepidermal layers showed little change, whereas the innermost layers exhibited the greatest increase. Electron microscopy revealed that ethylene increased both the rough endoplasmic reticulum and dictyosomes, suggesting that ethylene stimulated the secretion of cell wall materials. In untreated seedlings, the pattern of cell growth was similar in cells from the epidermis, cortex, and pith. The final cell size varied along the hypocotyl, the cells becoming shorter and broader the closer to the basal zones of the organ.

Thigmomorphogenesis: Changes in Cell Division and Elongation in the Internodes of Mechanically-perturbed or Ethrel-treated Bean Plants*

Examination of first internodes of young Phaseolus vulgaris L. plants which have been subjected to mechanical perturbation shows decreased elongation and increased radial growth. The decreased elongation can be attributed to both reduced cell elongation of epidermal and cortical cells and a reduced number of cells in the vascular and pith tissues. The increased radial enlargement is due to increased cortical cell expansion and increased secondary xylem production resulting from increased cambial activity. All of these responses are observable within a few hours of a single mechanical perturbation. Treatment of plants with ethrel mimics all of these effects of mechanical perturbation.

Wheat and Wild Oat Response to Flufenprop-methyl

The tolerance of wheat (Triticum aestivum L. ‘Waldron’) and wild oat (Avena fatua L.) to various rates of flufenprop-methyl {methyl-2-[benzoyl(3-chloro-4-fluorophenyl)amino]propanoate} applied weekly after wheat and wild oat emergence was determined under field conditions. Wild oat control increased at all growth stages as flufenprop-methyl rate increased. Wild oat control was greater than 80% with flufenprop-methyl at all rates when applied up to 6 weeks after wild oat emergence: (anthesis stage), but decreased when application was delayed further. Wheat was most susceptible to flufenprop-methyl during anthesis. Flufenprop-methyl at 0.56 kg/ha injured weed-free wheat only at the boot and anthesis stages. Injury intensity and the number of weeks that injury remained evident increased as flufenprop-methyl rate increased. Flufenprop-methyl injury to wheat was expressed as reduced plant height, grain yield, and kernels per spike and increased grain protein. Plant height reductions were attributed to reduced cell elongation. Grain yield reductions resulted from reduced kernels per spike.

Observations on chick-embryo lens histogenesis in vivo and in vitro.

The 6-day chick-embryo lens anterior epithelium will undergo histogenesis in vitro. A 'normal' epithelial curvature in vitro is required for extensive fiber-cell elongation; a reversed curvature, or flat epithelial conformation, results in considerably reduced cell extension. With a 'normal' epithelial curvature, cell elongation in vitro progresses rapidly for about 3 days, and changes in cytoplasmic organization (ultrastructure) take place that are similar to those associated with fiber-cell differentiation in vivo. However, these cells do not achieve as full a cytoplasmic differentiation as was observed in the most differentiated cells of the normal embryonic lens. Epithelia explanted with a reversed curvature, while evidencing reduced cell elongation, had, after 3 days in vitro, differentiated (according to criteria of ultrastructure and relative proportions of soluble proteins) in a manner similar to those epithelia explanted with a 'normal' curvature and in which extensive cell elongation had been demonstrated.

Morphogenetic studies on excised leaves of Osmunda cinnamomea: histological studies of leaf development in sterile nutrient culture

Leaf primordia of Osmunda cinnamomea L. explanted to a culture medium of simple composition at the end of three of their five seasons of development, complete their normal morphogenetic sequence but produce mature leaves of greatly reduced size. A histological study of the ground tissues of the rachis of cultured leaves reveals that, at least in these tissues, the size reduction results from a diminished cell number rather than from reduced cell elongation. It is apparent that, at every stage of development, cell division is greatly curtailed and that cell elongation begins precociously. Nevertheless, the pattern of development, including the distribution of mitotic activity at critical stages in the morphogenesis of the leaf, is normal. It is concluded that the effects of isolation upon leaves at this stage of development are quantitative ones upon the extent and duration of cell division and cell elongation.

THE TRANSITION FROM FILAMENTOUS TO TWO‐DIMENSIONAL GROWTH IN FERN GAMETOPHYTES. II. KINETIC STUDIES ON PTERIDIUM AQUILINUM

Gametophytes of the fern Pteridium aquilinum were incubated 3 days in red light and then transferred to white light. The sequence of events occurring after the transfer was as follows: a swelling of the apical region within 1 hr; a reduction in cell elongation after 5 hr; a series of one‐dimensional cell divisions between 10‐25 hr; and the initiation of two‐dimensional gametophytes after 25‐50 hr. The percentage of two‐dimensional gametophytes was proportional to the logarithm of the intensity of white light. The rate of elongation after 5 hr was inversely proportional to the logarithm of the intensity. The rates of cell divisions for one‐ and two‐dimensional gametophytes were proportional to intensity up to 240 and 120 ft‐c, respectively.