How periodic growth pattern and source/sink relations affect root growth in oak tree seedlings

Seedlings of Quercus pubescens were grown in root boxes to study the growth pattern of the root system in relation to shoot development. Shoot growth was typically rhythmic. Root elongation was also periodic, in contrast to several previous reports on other Quercus species. Both taproot and lateral root elongation were depressed during expansion of the second leaf flush, with a more pronounced response of lateral root growth. Apical diameter of the taproot followed comparable but less prominent trends than taproot elongation. Modifying source/sink relationships through various defoliation treatments altered the root growth pattern. Ablation of source organs (mature leaves or cotyledons) amplified the decrease in root growth concomitant with leaf expansion. Root growth recovery was even more difficult when both cotyledons and mature leaves had been removed. Ablation of sink aerial organs (young leaves) initially suppressed competition for growth between the shoot and the root, and then caused a gradual decrease in lateral root growth. Antagonism between maximum leaf expansion and root growth reduction during the second flush, and various responses of seedlings with modified source/sink relationships, raise an hypothesis of mutual competition for carbohydrates. The gradual decrease in lateral root growth after ablation of young leaves suggests a long-term carbohydrate limitation, or auxin limitation as auxin sources have been removed.     

Active, inactive and in vitro synthesized forms of polyphenoloxidase during leaf development

Polyphenoloxidase (PPO; EC 1.14.18.1) activity decreased 8-fold from young to mature Vicia faba L. Moensch (cv. Long pod) leaves. The K_m for catechol remained relatively constant from young to mature leaves. Electrophoretic separation and analysis showed that only one active form was present in extracts from various leaf sizes. The amount of this form appeared to decrease with leaf size/age. In extracts which had not tanned, electroblotting and immunostaining indicated that one enzyme form was present with a molecular mass of 45 kDa. Two leaf categories contained greater amounts of this immunological cross-reacting PPO than other leaf categories. When extracts were allowed to darken, immunoblotting detected three enzyme forms with molecular masses of 45, 59 and 63 kDa. The latter two immunological crossracting species had no detectable enzyme activity. Poly-A⁺ mRNA was isolated from six leaf sizes and translated in vitro. A product corresponding to PPO was present in all leaf categories. Greater amounts of this translation product were observed in medium-sized leaves than in very young or mature leaves. These results suggest that: (1) enzymatic assays for PPO are not reliable indicators of the total amount of PPO protein present in developing leaves, (2) immunoblotting can detect inactive enzyme forms, (3) only one active form of the enzyme is present at all developmental stages, and (4) mRNA corresponding to PPO is present at all developmental stages but appears to be more abundant in certain leaf sizes/ages.     

Biosynthesis of Branched-Chain Amino Acids in Schizosaccharomyces pombe: Properties of Acetohydroxy Acid Synthetase1

The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.     

Suboptimal temperature favors reserve formation in biennial carrot (Daucus carota) plants

In response to suboptimal temperatures, temperate annual plants often increase root:shoot ratios, build-up carbohydrates and display typical morphological and anatomical changes. We know less about the responses of biennials such as carrot. As a model plant, carrot has the additional feature of two functionally and morphologically distinct root parts: the taproot, which stores carbohydrate and other compounds, and the fibrous root system involved in acquisition of water and nutrients. Here, we analyze the effects of temperature (12 vs 25°C) on growth, carbohydrate accumulation and whole-plant morphology in two carrot cultivars. Our working hypothesis is that suboptimal temperature favors active formation of reserve structures, rather than passive accumulation of storage carbohydrates. In comparison with plants grown at 25°C, plants grown at 12°C had: (1) higher fibrous root:shoot ratio (13%) , (2) thicker (10–15%) and smaller (up to two- to three-fold) leaves, (3) lower leaf cuticular permeance (two- to four-fold), (4) higher taproot:shoot ratio (two-fold), (5) higher phloem:xylem ratios in taproot (two- to six-fold), (6) unchanged percentage dry matter content (%DMC) in leaves, petioles or fibrous roots and (7) higher %DMC in taproot (20%). However, %DMC of individual taproot tissues (phloem and xylem) was unaffected by temperatures and was consistently higher in the phloem (up to 30%). Therefore, the higher %DMC of whole taproots at 12°C was attributed solely to the increased development of phloem tissue. Carrot, therefore, shares many of the most conspicuous elements of temperate plant responses to low temperatures. Consistently with our hypothesis, however, carrots grown at suboptimal temperature promoted reserve structures, rather than the increase in carbohydrate concentration typical of most temperate annual species and woody perennials.     

Subunit association in acetohydroxy acid synthase isozyme III.

Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is composed of large and small subunits (encoded by the genes ilvI and ilvH) in an alpha 2 beta 2 structure. The large (61-kDa) subunit apparently contains the catalytic machinery of the enzyme, while the small (17-kDa) subunit is required for specific stabilization of the active conformation of the large subunit as well as for valine sensitivity. The interaction between subunits has been studied by using purified enzyme and extracts containing subcloned subunits. The association between large and small subunits is reversible, with a dissociation constant sufficiently high to have important experimental consequences: the activity of the enzyme shows a concentration dependence curve which is concave upward, and this dependence becomes linear upon the addition of excess large or small subunits. We estimate that at a concentration of 10(-7) M for each subunit (7 micrograms of enzyme ml-1), the large subunits are only half associated as the I2H2 active holoenzyme. This dissociation constant is high enough to cause underestimation of the activity of AHAS III in bacterial extracts. The true activity of this isozyme in extracts is observed in the presence of excess small subunits, which maintain the enzyme in its associated form. Reexamination of an E. coli K-12 ilvBN+ ilvIH+ strain grown in glucose indicates that AHAS III is the major isozyme expressed. As an excess of small subunits does not influence the apparent Ki for valine inhibition of the purified enzyme, it is likely that valine binds to and inhibits I2H2 rather than inducing dissociation. AHAS I and II seem to show a much lower tendency to dissociate than does AHAS III.     

Effects of Manganese Toxicity on the Growth and Gene Expression at the Seedling Stage of Soybean

In order to investigate the effects of Manganese (Mn) toxicity stress on the growth and gene expression at the seedling stage of soybean, soybean seedlings were treated with normal Mn concentration (5 μmol/L MnSO₄) and excess Mn concentration (100 μmol/L MnSO₄) by the method of hydroponic culture in this study. When soybean was subjected to Mn toxicity stress, excessive Mn could affect seedling growth, root development, the number of Mn oxide spots in leaves, and the Mn accumulation content in different parts of soybean. With the increase of exogenous Mn concentration and the prolongation of culture time, the shoot and root biomasses of soybean decreased significantly, but the plant height of soybean had no obvious effects. The total root length, root surface area and root volume of soybean decreased significantly, but the taproot length, taproot tip length and root diameter did not change significantly. The number of Mn spots in the primary leaves (first leaf) was significantly more than that in the old leaves (second leaf) and the youngest leaves (fourth leaf). The Mn concentration in leaves was significantly higher than that in roots, and the Mn concentration in the old leaves was significantly higher than that in youngest leaves with the method of inductively coupled plasma atomic emission spectrometry (ICP-AES). Moreover, the results in the present study suggested that the 10 selected genes were significantly up-regulated or down-regulated by Mn toxicity in the old and young leaves by quantitative real-time PCR (qRT-PCR) analysis. This indicates that these genes might be important in the process of regulation in old and young leaves of the physiological responses and ion transporting to Mn toxicity stress.     

ent-Kaurene Biosynthesis in Cell-Free Extracts of Excised Parts of Tall and Dwarf Pea Seedlings

Investigations on the sites of ent-kaur-16-ene (ent-kaurene) biosynthesis were conducted with cell-free extracts from several excised parts of 10-, 13-, and 16-d-old tall and dwarf pea (Pisum sativum L.) seedlings. [¹⁴C]Mevalonic acid was incorporated into ent-kaurene in cell-free extracts from young developing leaves and elongating internodes of tall (`Alaska') and dwarf (`Progress No.9') pea seedlings at all three stages of development. ent-Kaurene biosynthesis also occurred readily in cell-free extracts from shoot tips, petioles, and stipules near the young elongating internodes. The ent-kaurene-synthesizing activity found in young developing tissues declined as tissues matured. Little or no activity was detectable in enzyme extracts from cotyledons and root tips at different stages. In light grown tall pea internodes ent-kaurene-synthesizing activity was low as they began to elongate, reached a maximum when the internodes reached about 2 cm in length and declined as they matured. Activity in extracts of dwarf shoot tips and internodes was generally lower than in equivalent tall plants, but the activity in dwarf leaves and stipules was somewhat higher than in tall plants. With the exception of root tips, there is a strong correlation between growth potential of a tissue and the rate of ent-kaurene biosynthesis in extracts from that tissue.     

Comparative Enzymic Studies of Sucrose Metabolism in the Taproots and Fibrous Roots of Beta vulgaris L

Comparative enzymic studies of sugar beet (Beta vulgaris L.) taproots and fibrous roots revealed differences in invertase (EC 3.2.1.26) and sucrose synthetase (EC 2.4.1.13) activity. Invertase activity of the two root forms differs with respect to specific activity, pH optimum, and enzyme solubility. Acid invertase (pH 4.5) in the taproot was restricted to the peripheral meristematic tissue which produces cells for both taproot and fibrous root growth. This finding supports the hypothesis that the enzyme regulates sucrose partitioning between the taproot and fibrous roots. A distinct alkaline invertase (pH 8.0) was detected in sucrose storage tissues of the taproot.     

Nitrate Reductase in the Field Pea (Pisutn arvense L.)

The extraction and assay of a soluble, NADH-requiring nitratereductase is described. Induction of the enzyme by nitrate isdemonstrated in the roots and shoot of young seedlings and inthe leaves of older, nodulated plants grown without inorganicnitrogen. Induction occurs most readily in actively growingtissues. Studies with sterile cultures of excised roots confirmthe presence of an endogenous enzyme system within the root. Assays of the enzyme in vitro are combined with analyses offree nitrate and organic compounds of nitrogen in the bleedingsap. Either the root or shoot may act as the main centre forreduction of incoming nitrate. The extent to which these organsfunction is apparently influenced by environmental factors,particularly the level of nitrate in the rooting medium. Thediurnal rhythm of export of nitrogenous substances from theroot is correlated with observations on daily fluctuations inthe level of extractable enzyme in root and leaves. The activity of the enzyme is studied in different ages of leafof plants grown on a constant supply of nitrate. Assays suggestthat the enzyme is most active just as a leaf is fully expanded.Thereafter enzyme activity falls sharply, although small amountsof active enzyme may be recovered until a leaf becomes senescent.     

Competitiveness of Frankia strains on Elaeagnus clonal plantlets

Abstract Growth of the cyanobacterium Spirulina platensis , like that of many prokaryotic and eukaryotic organisms, is inhibited by low concentrations of valine, one of the three end-products of the branched-chain amino acid biosynthetic pathway. We assayed and partially characterized the activity of acetyhydroxy acid synthase (AHAS), the first common enzyme of the branched pathway in cell-free extracts from axenic S. platensis cultures. Assays performed at various pH values showed two peaks of activity, both inhibited by valine. FAD was not required for enzyme activity but protected it during dialysis. We also investigated whether the three amino acids were able to cause repression of AHAS synthesis and a significant drop in the enzyme-specific activity could be seen only when cultures were grown in the presence of valine. Chromatography on hydroxylapatite showed one single peak of activity.     

松嫩平原全叶马兰种群不同龄级分株的生长策略

全叶马兰为根蘖性多年生草本菊科植物,种群由3个龄级无性系分株组成,在松嫩平原6月份,是全叶马兰营养生长的旺盛期,研究结果表明,分株的生长与生产力以1龄级最低,2、3龄级依次增高,3个龄级分株的生物量分配均为茎>叶>主根,叶的生物量分配为1龄级>2龄级>3龄级,茎和主根的均为1龄级<2龄级<3龄级,3个龄级分株总重与分株高度、分株叶重与茎重、分株主根重与枝条重之间均为幂函数异速生长规律,主根的生物量分配则随着分株高度的增加呈线性减少,全叶马兰无性系分株的生长与分配策略是在分株幼小时将较多的物质分配给叶,以保证物质生产器官叶的建造上,随着分株生长逐渐将生产的物质分配到茎和主根的生长与贮藏;分株不向主根投入较多的能量,但在分株幼小时向主根中分配的物质相对较多,随着分株的生长则按比例较小,并保持相对稳定的分配比率     

Allometric relationships in young seedlings of faba bean (Vicia faba L.) following removal of certain root types

Sink-source relationships and allometric ratios were studied in young seedlings of faba bean (Vicia faba L.) following pruning of some root types. The plants were grown in an aeroponic system allowing an easy access to each part of the root system, throughout the experiment, without disturbing the others. Root, leaf and stem growth as well as their mineral content were determined in one group of undisturbed plants (CTRL) and in four groups of plants treated as follows: TAP – the distal-free portion of the taproot was removed; HALF – half the laterals were removed; ALL – all lateral roots were removed, and TAP+HALF – both the distal part of the taproot and half of the laterals were removed. Removal of all the lateral roots (ALL) induced the development of a longer taproot but severely arrested shoot growth. Phosphorus, potassium and calcium contents were lower in the plants of the ALL treatment. However, the content of Mg was practically unaffected. The effect of the HALF treatment was hardly noticeable but the effects of TAP+HALF treatment were cumulative. The allometric relationships between the surface area of the roots and that of the leaves were restored within the experimental period, apparently due to reduction in shoot growth. Removal of the distal parts of the taproot did not cause an increase in shoot growth. This indicates that the strength of the sinks (mostly of lateral roots) rather than that of the source determines these relationships.     

Modification of root architecture in woody plants is possible for the presence of two different mechanisms of lateral root production: The effect of slope in Spartium junceum L. seedlings

Abstract

This paper investigates the modification of root architecture of Spartium junceum L. seedlings grown in slope condition. It is reported that 50% of the total number of lateral roots are concentrated in few centimetres of the taproot near the collar. The anatomical analysis of transverse sections along the taproot axis reveals that this taproot zone is characterised by two types of lateral roots: one with a trace extending to the centre of the vascular cylinder by following the path of a medullar ray; one with a trace which ends in the vascular cambium. The first type may be lateral roots originated from the taproot primary structure; the second type seems to be lateral roots developing later when a secondary structure has completely substituted the primary structure. The emission of this second type of lateral roots seems to be strongly controlled by environmental conditions with considerable consequences upon the overall root architecture. In the example reported in this paper, young plants growing under mechanical stress due to a slope develop asymmetric root architecture with lateral roots elongating in two prevalent directions: up-slope or down-slope. This asymmetric architecture is produced in the zone of the taproot where a secondary structure is present and represents the plant response to the need of increasing its anchorage strength.     

The Relationship between Cyanide-Resistant Root Respiration and the Storage of Sugars in the Taproot in Daucus carota L.

Dry weight increase, root respiration, photosynthesis, and shootdark respiration were measured prior to and during the developmentof the taproot of carrot (Daucus carota L., var. AmsterdamseBak) which stores sucrose and reducing sugars. Before storageof sugars in the taproot started, root respiration was highand up to 50% inhibited by salicyl hydroxamic acid (SHAM), indicatinga high activity of the alternative pathway. The onset of sugar storage in the taproot coincided with a sharpdecrease in alternative pathway activity. When sugars were storedin the taproot, the alternative pathway in the taproot was notoperative, although present. It was concluded that the alternative pathway only consumessugars that are not used for energy production in growth ormaintenance processes, for carbon skeletons, osmoregulationor storage, in accordance with the energy overflow model. Apositive quantitative relationship exists between the storageof sugars in the taproot and the efficiency of root respiration.After day 32 the amount of daily produced carbohydrates transportedto the root system increased and these sugars were stored inthe taproot. In this period less sugars were invested in structuralshoot growth. In the present investigation no positive influence from thedeveloping sink is found on photosynthetic rate or net assimilationrate. Both rates decrease with increasing age of the plants,although increasing amounts of sugars were stored in the taproot.     

Acetohydroxy Acid Synthase Activity in Chlorella emersonii under Auto- and Heterotrophic Growth Conditions

Acetohydroxyacid synthase (AHAS) activity was studied in the green unicellular alga Chlorella emersonii. This activity and its regulation was compared in the algae grown autotrophically and heterotrophically on glucose in the dark. No evidence for the existence of more than one enzyme was found. The activity in crude extracts from either heterotrophically or autotrophically grown cells showed a K_m for pyruvate of 9 millimolar, a 22-fold preference for 2-ketobutyrate over pyruvate as the second substrate, 50% inhibition by 0.5 millimolar valine, and 50% inhibition by 0.3 micromolar sulfometuron methyl (SMM). Spontaneous mutants of the alga resistant to SMM were isolated, which appeared to be single gene mutants containing SMM-resistant AHAS activity. Hence, AHAS appears to be the sole direct target site of SMM in C. emersonii. The fact that the mutants had equivalent SMM resistance under auto- and heterotrophic conditions further supports the conclusion that the same enzyme functions under both physiological regimes. The addition of valine and isoleucine leads to partial relief of SMM inhibition of biomass increase, but not of SMM inhibition of cell division.     

Regulatory effects of exogenous branched-chain amino acids in Nicotiana plumbaginifolia cell suspension cultures

Nicotiana plumbaginifolia suspension cultured cells were grown on medium supplemented with valine, leucine and isoleucine, singly or in combination. The effects of the three branched-chain amino acids on cell growth rate and on the activity of acetohydroxyacid synthase (AHAS), the first enzyme (and the main regulative site) of their biosynthetic pathway, were studied. Results showed that valine and leucine, at concentrations ranging from 10^–4 to 10^–3 M, inhibit growth, and at higher doses (from 10^–2 to 10^–1 M) AHAS activity. Growth, but not AHAS activity, was affected also by isoleucine. The addition of ammonium succinate to the culture medium, in order to counteract a possible general inhibitory effect of these compounds on nitrogen metabolism, relieved only partially their cytotoxicity. Feeding cells with equimolar mixtures of the three amino acids resulted in a minor but reproducible decrease in AHAS level, which was proportional to the dose. A similar result was obtained also on N. plumbaginifolia seedlings, suggesting that in this species a modulation of enzyme level could play a role in controlling the flow of metabolites through the pathway.Abbreviations AHAS acetohydroxyacid synthase - BCAA branched-chain amino acids - FAD flavin adenine dinucleotide - GS glutamine synthetase - TPP thiamine pyrophosphate     

Increase in glutamate synthase (NADH) activity in maize seedlings in response to nitrate and ammonium nitrogen

Roots and leaves of Zea mays L. cv. Ganga Safed-2 seedlings grown with nutrient solution containing either 10 m M KNO₃ or NH₄Cl or 5 m M NH₄NO₃ had considerably higher glutamate synthase (NADH, EC 1.4.1.14) activity than the corresponding organs from seedlings grown without any nitrogen. The supply of inorganic nitrogen for a short time, i.e. 3 h, to roots and leaves excised from seedlings grown without nitrogen also increased the enzyme activity in these organs. This increase was more pronounced with nitrate than with ammonium nitrogen. When excised roots and leaves from NH₄NO₃-grown seedlings were incubated in a minus nitrogen medium for 24 h, the enzyme activity declined considerably. This decline was inhibited to some extent by nitrogen, especially by nitrate. Inorganic nitrogen prevented similarly the decline in in vitro enzyme activity during 24 h storage at 25°C, more regularly for the root than for the leaf enzyme. The experiments demonstrate the role of inorganic nitrogen in the regulation of glutamate synthase activity.     

Physical and kinetic properties of acetohydroxyacid synthase from wheat leaves

Acetohydroxyacid synthase (AHAS, EC 4.1.3.18; also known as acetolactate synthase), which catalyses the first reaction common to the biosynthesis of the branched-chain amino acids, L-valine, L-leucine and L-isoleucine, and is the target of several classes of herbicides, has been studied in hydroponically-grown seedlings of wheat (Triticum aestivum L. cv. Vulcan). Enzyme activity was greater in leaves than roots, reaching a maximum between 4 and 6 days after germination. AHAS was associated with the chloroplasts after centrifugation in a density gradient. A preparation of the enzyme was obtained from wheat leaves which gave a single band after electrophoresis in native gels but was resolved by denaturing sodium dodecyl sulphate-polyacrylamide gel electrophoresis into three polypeptide bands of molecular mass 58, 57 and 15 kDa. The native molecular mass was approximately 128 kDa. AHAS had optimum activity at pH 7 and did not require the addition of flavin adenine dinucleotide (FAD), thiamine pyrophosphate (TPP) and MgCl₂ for activity. The enzyme did not display typical hyperbolic kinetics, in that the double reciprocal plot of activity against pyruvate concentration was non-linear. The concentration of pyruvate that gave half of the maximum activity was 4 mM. Sulfonylurea and imidazolinone herbicides were potent inhibitors of wheat leaf AHAS, with 50% inhibition being observed at concentrations of 0.6 and 0.3 μM for chlorsulfuron and metsulfuron methyl, respectively, and at 2.5, 5 and 10 μM for imazaquin, imazethapyr and imazapyr. Inhibition by both classes of compounds was reversed by removal of the inhibitor. Progress curves of product formation against time in the presence of the herbicides were non-linear, and based on the assumption that inhibition by the sulfonylureas was of the slow, tight-binding type, estimates of 0.17 and 0.1 nM were obtained for the dissociation constants of chlorsulfuron and metsulfuron methyl, respectively, from the steady-state enzyme-inhibitor complex.     

Correlation of Pectolytic Enzyme Activity with the Programmed Release of Cells from Root Caps of Pea (Pisum sativum)

In many plant species, the daily release of hundreds to thousands of healthy cells from the root cap into the soil is a normal process, whose function is unknown. We studied the separation of the cells in pea (Pisum sativum) using an aeroponic system in which separated cells were retained on the root until they were washed off for counting. We found that cell separation is a developmentally regulated, temperature-sensitive process that appears to be regulated independently of root growth. No cells were released from very young roots. When plants were grown aeroponically, cell numbers increased with increasing root length to a mean of 3400 cells per root, at which point the release of new cells ceased. The process could be reset and synchronized by washing the root in water to remove shed cells. Cell separation from the root cap was correlated with pectolytic enzyme activity in root cap tissue. Because these cells that separate from the root cap ensheath the root as it grows and thus provide a cellular interface between the root surface and the soil, we propose to call the cells “root border cells.”