Cellular Growth in Roots of a Gibberellin-Deficient Mutant of Tomato (Lycopersicon esculentum Mill.) and its Wild-type

The role of gibberellins in regulating the growth of tomatoroots was investigated by comparing various cellular parametersin cultured roots of the gibberellin-deficient mutant gib-l/gib-lwith those in roots of the near-isogenic wild-type. In addition,wild-type roots treated with 0?1 µM 2S,3S paclobutrazol,an inhibitor of gibberellin biosynthesis, and mutant roots treatedwith 0?1 µM GA₃ were also compared: the former roots constitutea phenocopy of the mutant, whereas the latter roots appear tobe ‘normalized’ and similar to wild-type. The elongationof mutant and phenocopied roots were similar, their maximumelongation rates being about half or two-thirds that of wild-typeor GA₃-treated mutant roots, respectively. These rates wereinterpreted in terms of the numbers and lengths of cells withinthe meristematic and non-meristematic portions of the elongationzone. Mean meristem length tended to be shorter in both themutant and the 2S,3S paclobutrazol-treated wild-type roots thanin the other two types of root. A major difference between thetwo pairs of mutant and normal roots was their mean final celllengths: mean lengths of cortical cells of the mutant and 2S,3Spaclobutrazol-treated roots were, respectively, 39% and 25%shorter than the mean length of wild-type roots. Final celllength in the GA³-treated mutant roots were similar to wild-type.By contrast, the diameters of mature cortical cells of the mutantand phenocopy were about 20% greater than the diameters of equivalentwild-type or ‘normalized’ mutant cells. The meanvolumes of cortical cells in all four types of roots showedno significant differences. Knowledge of the distribution ofcortical cell lengths, widths and volumes along the root axis,together with information about the rate of root elongation,permitted comparisons of the relative elemental growth ratesof each of these three cellular parameters. The available evidence suggests that the level of endogenousgibberellins in mutant roots is lower than in wild-type roots.The present results, therefore, suggest that endogenous gibberellinsare necessary for normal growth of cultured tomato roots andthat they regulate the relative amounts of growth at the longitudinaland transverse walls of the cells which, in turn, affects theshape of the elongating cells. Key words: Cell growth, cultured roots, gibberellin, gib-l mutant, Lycopersicon esculentum, 2S,3S paclobutrazol, relative elemental growth rate, root meristem     

Effect of Root and Shoot Meristem Temperature on Shoot to Root Dry Matter Partitioning and the Internal Concentrations of Nitrogen and Carbohydrates in Maize and Wheat

Maize (Zea mays L.) and spring wheat (Triticum aestivum L.)were grown in nutrient solution at uniformly high air temperature(20 °C), but different root zone temperatures (RZT 20, 16,12 °C). To manipulate the ratio of shoot activity to rootactivity, the plants were grown with their shoot base includingthe apical meristem either above (i.e. at 20 °C) or withinthe nutrient solution (i.e. at 20, 16 or 12 °C). In wheat, the ratio of shoot:root dry matter partitioning decreasedat low RZT, whereas the opposite was true for maize. In bothspecies, dry matter partitioning to the shoot was one-sidedlyincreased when the shoot base temperature, and thus shoot activity,were increased at low RZT. The concentrations of non-structuralcarbohydrates (NSC) in the shoots and roots were higher at lowin comparison to high RZT in both species, irrespective of theshoot base temperature. The concentrations of nitrogen (N) inthe shoot and root fresh matter also increased at low RZT withthe exception of maize grown at 12 °C RZT and 20 °Cshoot base temperature. The ratio of NSC:N was increased inboth species at low RZT. However this ratio was negatively correlatedwith the ratio of shoot:root dry matter partitioning in wheat,but positively correlated in maize. It is suggested that dry matter partitioning between shoot androots at low RZT is not causally related to the internal nitrogenor carbohydrate status of the plants. Furthermore, balancedactivity between shoot and roots is maintained by adaptationsin specific shoot and root activity, rather than by an alteredratio of biomass allocation between shoot and roots.Copyright1994, 1999 Academic Press Wheat, Triticum aestivum, maize, Zea mays, root temperature, shoot meristem temperature, biomass allocation, shoot:root ratio, carbohydrate status, nitrogen status, functional equilibrium     

Structural Changes During the in vitro Germination of Vanilla planifolia (Orchidaceae)

A study is reported of histogenesis and organogenesis duringthe processes leading up to seedling formation in cultures ofVanilla planifolia. Prior to germination, all cells of the embryoincreased in size rupturing the seed coat and initiating theprotocorm stage. The cells of the protocorm were heavily ladenwith starch grains. Although all of the cells of the matureembryo were heavily laden with protein bodies, these were confinedto the terminal cell descendents on emergence of the embryofrom the seed coat, and they disappeared during differentiationof the meristem, indicating that some reserves were mobilizedand utilized during germination. The terminal locus of embryonal axis did not differentiate intoa cotyledon and epicotyl as in other angiosperm embryos butformed a thick meristematic layer. Bipolar differentiation withinthe meristem produced the shoot, and after a few leaves hadbeen formed, the first root differentiated endogenously fromthe base of the meristem. Subsequent roots, however, appearedto originate more superficially. The chain of events appearsto be quite unique to Vanilla amongst the angiosperms. Vanilla planifolia, protocorm, starch grains, protein bodies     

The Meristem and Quiescent Centre in Cultured Root Apices of the gib-l Mutant of Tomato (Lycopersicon esculentum Mill.)

Cultured root apices of tomato bearing the gib-I mutation, whichreduces the levels of endogenous gibberellins, grew slower andwere thicker than wild-type contols. This was the result ofshorter and broader cells in the menstem of the mutant. Cellsof both cortex and stele were affected, but this did not causeany alteration to the volume fraction occupied by these twotissues in the root meristem. Root caps were longer in the mutantand there were also more layers of rhizodermis. All these effectscould be reproduced in wild-type roots by addition of 0.1µM2S, 3S paclobutrazol (an inhibitor of gibberellin biosynthesis)to the culture medium and could be normalized in mutant rootsby 0.1 µM GA₃. Cell doubling times in the proximal regionof the meristem were similar in mutant and wild-type roots,but were faster in both the quiescent centre (QC) and the capmeristem of the mutant. This latter feature of the mutant rootsis likely to be the cause of their longer caps, while the fasterrate of division in the QC accounts for the additional tiersof cells that were found to build up in the cortical portionof this zone These additional tiers failed to form in mutantroots grown in GA₃, but they could be induced in wild-type rootsby 2S, 3S paclobutrazol. These results suggest that endogenousgibberellins may be partly responsible for the slow rate ofcell growth and proliferation in the QC. Gibberellins, gib-I mutation, Lycopersicon esculentum, meristem, roots, 2S, 3S paclobutrazol, quiescent centre, tomato     

In Vitro Transformation of Root meristem to Shoot and Plantlets in Vanilla planifolia

A study is reported of histogenesis and organogenesis duringthe processes leading up to plantlet formation in tip culturesof aerial roots of Vanilla planifolia. Young root tips excisedfrom aerial roots, less than 15 cm long, when cultured in liquidMS medium containing IAA and KN showed gravitropic responseuntil cap lysis began. With the collapse of the distal halfof the cap, the cells of the quiescent centre divided forminga hemispherical mass of cells. Further localized divisions onthe periphery of the hemisphere resulted in a number of meristemoidseach of which differentiated into a shoot meristem with leafprimordia. Procambium differentiated first beneath the apicalmeristem after two to three leaf primordia had formed and thenat the base of the leaves. After a few leaves have been formeda root meristem differentiated in close lateral proximity tothe basal end of the shoot procambium. Formation of a plateof vasculature at the nodal region of the first formed leaf,procambialization of the root and the bridging up of the shootand root vasculature with the nodal plate are described. Vanilla planifolia, root tip, in vitro, quiescent centre, meristemoid, plantlet     

Evidence for proteins specific for vascular elements in intact and cultured tissues and cells of maize

A group of antigenically distinct proteins characteristic for the tissue complex of the vascular cylinders was found in maize (Zea mays L.) seedlings using an immunofiltration technique. Specific stelar antigens present in the fully developed stele (vascular cylinder) of the primary root were also found in steles extracted from adventitious roots and from the mesocotyl but were absent, within the limits of sensitivity of the immunodiffusion tests employed, in root cortex and epidermis. Some of the stelar antigens were also evident in the meristem of the primary root and were present in traces in the scutellum, the mesocotyl node, and the primary leaves plus coleoptile. The specific stelar antigens could be traced in 13- and 15-day-old developing embryos and were definitely expressed by the 21 st day after pollination. Several stelar-specific antigens were found in embryo-derived callus tissues and in stem-derived cells maintained in serial suspension culture. Higher resolution of the stelar antigens by a modified technique of crossed immunoelectrophoresis was used to demonstrate several minor stelar antigens that were presumably characteristic exclusively of the completely differentiated stele. This technique along with sequential immunoprecipitation of labelled proteins provided a semiquantitative estimate of the specific stelar antigens in the meristem and the stele of the primary root, and in suspension-cultured cells which were devoid of noticeable signs of vascular differentiation.     

Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny.

Summary In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1–2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carring the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.     

Morphogenetic Aspects of a Leafless Mutant in Tomato: III. GROWTH AND PEROXIDASE ACTIVITY OF ROOTS

Using morphological and biochemical criteria, comparisons weremade between intact and excised roots of normal tomato (Lycopersiconesculentum Mill.) and the reduced form of the homozygous lanceolatemutant. Intact normal roots showed greater growth as reflectedin length of the main root axis, number of lateral roots, andprotodermal cell size. Excised normal roots grew more rapidlythan those of the mutant only during the first 24-h intervalof the first week in culture. Intact mutant roots revealed agreater activity of peroxidase, but excised mutant roots showedno increase in enzyme activity. It is concluded that the primarysite of action of the mutant allele is the shoot system, andin particular the leaf marginal meristem. The effects of thelanceolate gene on the root system in tomato are interpretedas being of secondary importance with regard to gene action.     

DRL1 regulates adaxial leaf patterning and shoot apical meristem activity in<Emphasis Type="Italic">Arabidopsis</Emphasis>

Leaf shape is controlled early on by initiation at the shoot apical meristem (SAM), as well as by changes in the rates and planes of cell division and the polarity-dependent differentiation of leaf cells. To elucidate the regulation of this differentiation by signal(s) from the SAM, we screened for mutations in genes that might be involved in these early processes. A novel recessive mutant, 356-2 [identified as a new allele of thedeformed root and leaf1 (drl1) mutant], was isolated from a collection ofDs transposon insertion lines. The356- 2/drl1- 101 mutant produces narrow, filamentous leaves and defective mer-istems. Its palisade cells have a spongy cell-like structure and are fewer in number, indicating that the leaves are abaxialized. Interestingly, some of those filament-like leaves have no vascular tissues inside their blades.DRL1 encodes a protein similar to the yeast elongator-associated protein (EAP) KTI12. The amino acid sequence of DRL1 is universally conserved in prokaryotes and eukaryotes. These facts suggest that DRL1 might positively regulate leaf polarity and SAM activity by controlling cell proliferation and differentiation.     

Comparative developmental anatomy of seedlings in nine species of podostemaceae (subfamily Podostemoideae)

The developmental anatomy is described for seedlings of nine Asian and Australian species of Podostemaceae, subfamily Podostemoideae. The hypocotyl is rudimentary (except in Zeylanidium olivaceum) and does not form a primary root in any of the species examined. An adventitious root forms endogenously in the hypocotyl of six species with ribbon-like or flattened subcylindrical roots, and in Z olivaceum with foliose roots. In contrast, it forms exogenously in Hydrobryum griffithii and Synstylis micranthera with foliose roots. The juvenile root becomes flattened and dorsiventral, branches exogenously (in Polypleurum stylosum, P. wallichii and Z. lichenoides) and produces shoots endogenously (in P. stylosum, P. wallichii, S. micranthera and Z. lichenoides). The root meristem is simple, composed of surface and uniform inner cells, and is devoid of root cap initials in all species. The reduced meristem morphology of seedling roots may be primitive in the Asian-Australian Podostemoideae. A root cap or protective tissue did not form during the culture period, even in the seven species with capped adult roots, probably due to its delayed development. It was absent throughout ontogeny in the other two species. No obvious shoot apical meristem forms between the cotyledons. One to several leaves occupy the shoot apical area in species with endogenous adventitious roots, while no leaves are formed in species with exogenous roots. These differences suggest recurrent origins of foliose roots in the Asian clade. Similarities between the unique seedling morphology and mutant Arabidopsis phenotypes are discussed.     

Seedling developmental anatomy of an undescribed Malaccotristicha species (Podostemaceae, subfamily Tristichoideae) with implications for body plan evolution

The seedling development of an undescribed Malaccotristicha species was observed by using seedling culture and microtomy to infer the evolution of body plan with a focus on the root, which is a developmentally leading organ of most Podostemaceae. The young seedling has a small primary shoot apical meristem and a primary root apical meristem. The shoot meristem develops into a plumular ramulus, and the root meristem, into a cylindrical radicle with no root cap. The radicle transforms to a dorsiventral, flattened, capped primary root. An adventitious root develops endogenously on the lateral side of the hypocotyl and is similar to the primary root. This is a new pattern in Podostemaceae. Comparison of this and described patterns of Podostemaceae (and the sister-group Hypericaceae) suggests that the radicle was lost in the early evolution of Podostemaceae and instead adventitious roots replaced it as a leading organ.     

An Early Cytochemical Marker of Commitment to Stelar Differentiation in Meristems from Dicotyledonous Plants

A cytochemical study of root apices from Vicia faba and Pisumsativum showed esterase activity to be present in the stele,root cap and rhizodermis, but almost completely absent fromthe developing cortex and quiescent centres. The meristem cellsgiving rise to the cortex were almost negative whilst thosegiving rise to the stele were positive for esterase activity.Cambia from roots, shoots and petioles of a number of dicotyledonousspecies were all positive for esterase activity. It is proposedthat esterase activity may be used as an early marker of commitmentto differentiation into stele in roots of dicotyledonous plants,and that the cambia are fully committed meristems. Pisum sativum L., Vicia fabaL., garden pea, broad bean, meristems, stelar differentiation, esterase activity, xylem differentiation, cytochemistry, cambium     

Genetic analysis of adventitious root formation with a novel series of temperature-sensitive mutants of Arabidopsis thaliana

When cultured on media containing the plant growth regulator auxin, hypocotyl explants of Arabidopsis thaliana generate adventitious roots. As a first step to investigate the genetic basis of adventitious organogenesis in plants, we isolated nine temperature-sensitive mutants defective in various stages in the formation of adventitious roots: five root initiation defective (rid1 to rid5) mutants failed to initiate the formation of root primordia; in one root primordium defective (rpd1) mutant, the development of root primordia was arrested; three root growth defective (rgd1, rgd2, and rgd3) mutants were defective in root growth after the establishment of the root apical meristem. The temperature sensitivity of callus formation and lateral root formation revealed further distinctions between the isolated mutants. The rid1 mutant was specifically defective in the reinitiation of cell proliferation from hypocotyl explants, while the rid2 mutant was also defective in the reinitiation of cell proliferation from root explants. These two mutants also exhibited abnormalities in the formation of the root apical meristem when lateral roots were induced at the restrictive temperature. The rgd1 and rgd2 mutants were deficient in root and callus growth, whereas the rgd3 mutation specifically affected root growth. The rid5 mutant required higher auxin concentrations for rooting at the restrictive temperature, implying a deficiency in auxin signaling. The rid5 phenotype was found to result from a mutation in the MOR1/GEM1 gene encoding a microtubule-associated protein. These findings about the rid5 mutant suggest a possible function of the microtubule system in auxin response.     

Experimental Modification of Cell Division Patterns in the Root Meristem of Zea mays

In the meristem of the young primary root of maize seedlingsthe first transverse division in the cortex 250 µm fromthe root apex results in two daughter cells of distinctly unequalsize. This division could be rendered equal by raising the seedlingsin up to 7.5% methanol. The pattern of the subsequent two orthree transverse divisions in the cortex, as revealed by thearrangement of the newly divided cells in the resultant cellularpackets, was acropetal in the methanol-treated roots but basipetalin the control roots. The sequence of division within a cellularpacket tended to follow the distribution of cell sizes - largercells divided earlier than smaller cells. A temporary arrestof cell division by exposing roots to cold (5 °C) conditionshad no effect on the sequence of divisions that followed whenthe roots were allowed to recover at 20 °C. The resultssuggest that the normally asymmetric position of the cell wallformed at cytokinesis is subject to active regulation and thatmethanol interferes with this process. The cytoplasm of certaincells in the root meristem was also found to be unequally distributed,as judged by Azure B staining, between the two ends of the cell.Cytoplasmic asymmetry was not directly correlated with inequalityof division, although it too was affected by methanol. Cell polarity, root meristem, unequal division, Zea mays     

Root Growth, Secondary Root Formation and Root Gravitropism in Carotenoid-deficient Seedlings of Zea mays L.

The effect of ABA on root growth, secondary-root formation androot gravitropism in seedlings of Zea mays was investigatedby using Fluridone-treated seedlings and a viviparous mutant,both of which lack carotenoids and ABA. Primary roots of seedlingsgrown in the presence of Fluridone grew significantly slowerthan those of control (i.e. untreated) roots. Elongation ofFluridone-treated roots was inhibited significantly by the exogenousapplication of 1 mM ABA. Exogenous application of 1 µMand 1 nM ABA had either no effect or only a slight stimulatoryeffect on root elongation, depending on the method of application.The absence of ABA in Fluridone-treated plants was not an importantfactor in secondary-root formation in seedlings less than 9–10d old. However, ABA may suppress secondary-root formation inolder seedlings, since 11-d-old control seedlings had significantlyfewer secondary roots than Fluridone-treated seedlings. Rootsof Fluridone-treated and control seedlings were graviresponsive.Similar data were obtained for vp-9 mutants of Z. mays, whichare phenotypically identical to Fluridone-treated seedlings.These results indicate that ABA is necessary for neither secondary-rootformation nor for positive gravitropism by primary roots. Zea mays, gravitropism, carotenoid-deficient, Fluridone, root growth, vp-9 mutant     

Gibberellins and the Growth of Excised Tomato Roots: Comparison of gib-1 Mutant and Wild Type and Responses to Applied GA3 and 2S, 3S Paclobutrazol

Clones of excised roots of wild type tomato (Lycopersicon esculentum,Mill., cv. Moneymaker) and a near-isogenic GA-deficient mutant(gib-1/gib-1) were cultured in modified White's medium containing1.5% w/v sucrose. The linear elongation rate of the main axisof the gib-1 mutant was 40% less than that of the wild type.In addition, the main axis of the gib-1 mutant was thicker thanthat of the wild type but main axis volume growth was the samein both genotypes, indicating that the gib-1 allele was affectingthe orientation of root expansion. There was no evidence tosuggest that the gib-1 allele affected either the pattern ofemergence or the density of lateral roots. Elongation rate andthickness of gib-1 mutant roots were restored to those of thewild type by the addition of low concentrations (0.1–1.0µM) of gibberellic acid (GA3). These concentrations ofGA3 caused a slight reduction in extension growth of wild typeroots, indicating that endogenous GAs were not limiting elongationof normal roots in culture. The GA biosynthesis inhibitor, 2S,3S paclobutrazol, at 0.1 µM, significantly reduced elongationof wild type roots and this inhibition was counteracted by 0.1µM GA₃. It is concluded that the difference in growthbetween the gib-1 mutant and the wild type represented GA-dependentgrowth. Low concentrations of 2S, 3S paclobutrazol caused onlya small (5%) reduction in growth of the gib-1 mutant and thisgrowth inhibition was not reversed by GA₃. This observation,and the fact that gib-1 mutant roots grow in the absence ofadded GA₃, suggested that part of root growth was GA-independent.However, the possibilities that the gib-1 mutant is ‘leaky’and that paclobutrazol does not inhibit GA biosynthesis completelycannot be excluded. Key words: gib-1 mutant, gibberellic acid, Lycopersicon esculentum, 2S, 3S paclobutrazol, root growth     

Lateral Root Anlage Development in Excised Roots of Vicia faba L., Pisum sativum L., Zea mays L. and Phaseolus vulgaris L.

The development of lateral root primordia has been investigatedin excised roots of Vicia faba, Pisum sativum, Zea mays andPhaseolus vulgaris cultured in White's medium supplemented with2 per cent sucrose and compared with previously published dataon such development in primaries of the corresponding intactplants (control roots). Primordia were produced in each batchof excised roots over the 6 day culture period but at a lowerrate (number day^–1) than in the controls. Such primordia in cultured roots of Zea and Phaseolus completedtheir development and grew out as lateral roots over a periodsimilar in length to that found in the controls, but with acell number of only about 33 per cent of that attained at thetime of secondary emergence in the primaries of the latter roots.These lower cell numbers were at least partly a reflection ofincreases in mean cell doubling time over the period of anlagedevelopment investigated in the excised roots relative to thecorresponding values found in the controls. Primordia initiated in excised roots of Pisum and Vicia didnot complete their development in culture, i.e. no lateral rootsemerged and arrest took place with cell numbers of only 37 (Pisum)and 17 (Vicia) per cent of the numbers determined at the timeof secondary root emergence in the controls. Such arrested primordiahad few nuclei in S and none in mitosis. Moreover, at leastin Pisum, the frequency distribution of the relative DNA contentof the nuclei in the latter primordia approximated that foundin the apical meristem of primary roots following the establishmentof the stationary phase under conditions of carbohydrate starvation. It has also been demonstrated in the course of these investigationsthat lateral root primordium development in all four speciesis at least biphasic and possibly triphasic. Vicia faba L., broad bean, Pisum sativum L., garden pea, Zea mays L., maize, Phaseolus vulgaris L., dwarf bean, root primordia, anlage, cell doubling time, lateral root emergence     

Developmental Morphology of Roots and root-borne shoots ofPodostemum subulatum as compared withZeylanidium olivaceum (Podostemaceae—Podostemoideae) Part VII of the series ‘Morphology of Podostemaceae’

The root structure ofPodostemum subulatum is investigated and compared with that ofZeylanidium olivaceum. Podostemum has thread- or ribbon-like roots. The root tip consists of an inner apical meristem and a single-layered root cap. From roots arise numerous shoots of endogeneous origin. Their vascular bundle isab initio connected with the root bundle.By the simple (reduced) apical zonation, the roots ofPodostemum subulatum appear more advanced than the crustose roots ofZeylanidium olivaceum, which bear an ordinary (though asymmetrical) root cap. With regard to the endogeneous root-borne shoots, however,Zeylanidium appears more advanced because of the shoot dimorphism. The floriferous shoots have a short axis that grows plagiotropously above the crust surface, whereas the axes of the vegetative shoots are extremely short and remain, together with the apical meristem, within the crust. Only the leaves protrude from the crust surface.