Localization of enzymes of assimilatory sulfate reduction in pea roots

The localization of enzymes of assimilatory sulfate reduction was examined in roots of 5-d-old pea (Pisum sativum L.) seedlings. During an 8-h period, roots of intact plants incorporated more label from ³⁵SO ₄ ^2- in the nutrient solution into the amino-acid and protein fractions than shoots. Excised roots and roots of intact plants assimilated comparable amounts of radioactivity from ³⁵SO ₄ ^2- into the amino-acid and protein fractions during a 1-h period, demonstrating that roots of pea seedlings at this stage of development were not completely dependent on the shoots for reduced sulfur compounds. Indeed, these roots contained activities of ATP-sulfurylase (EC 2.7.7.4), adenosine 5-phosphosulfate sulfotransferase, sulfite reductase (EC 1.8.7.1) and O-acetyl-l-serine sulfhydrylase (EC 4.2.99.8) at levels of 50, 30, 120 and 100%, respectively, of that in shoots. Most of the extractable activity of adenosine 5-phosphosulfate sulfotransferase was detected in the first centimeter of the root tip. Using sucrose density gradients for organelle separation from this part of the root showed that almost 40% of the activity of ATP-sulfurylase, adenosine 5-phosphosulfate sulfotransferase and sulfite reductase banded with the marker enzyme for proplastids, whereas only approximately 7% of O-acetyl-l-serine sulfhydrylase activity was detected in these fractions. Because their distributions on the gradients were very similar to that of nitrite reductase, a proplastid enzyme, it is concluded that ATP-sulfurylase, adenosine 5-phosphosulfate sulfotransferase and sulfite reductase are also exclusively or almost exclusively localized in the proplastids of pea roots. O-Acetyl-l-serine sulfhydrylase is predominantly present in the cytoplasm.Abbreviation APSSTase adenosine 5-phosphosulfate sulfotransferase     

Grain filling pattern and cytokinin content in the grains and roots of rice plants

Grain filling patterns and their relationships withzeatin (Z), zeatin riboside (ZR), indole-3-acetic acid(IAA) and gibberellin (GA) contents in the grains androots during grain development were examined in sixrice (Oryza sativa L.) genotypes grown in thefield and in water culture. Three grain fillingpatterns based on the filling rate of superior andinferior spikelets were observed, i.e., fastsynchronous: all spikelets started filling early andfast at the early filling stage; slowsynchronous: all spikelets filled slowly at the earlyfilling stage and reached the maximum filling ratelate; and asynchronous: superior spikeletsstarted filling and reached the maximum filling ratemuch earlier than the inferior ones. The order ofgrain filling percentage in the three types of grainfilling patterns was: fast synchronous >asynchronous > slow synchronous. Changes in Z + ZRcontents in the superior and inferior spikelets wereassociated with the grain filling patterns. Grainfilling percentage was significantly correlated withZ + ZR contents in the grains and roots at the earlyand middle grain filling stages. IAA and GA(GA¹ + GA³ + GA⁴)contents in the grains and roots were notsignificantly correlated with grain fillingpercentage. The results suggest that cytokinins in thegrains and roots during the early phase of graindevelopment play an important role in regulating grainfilling pattern and consequently influence grainfilling percentage.     

Phosphofructokinase of carrot roots

Phosphofructokinase was partially purified from carrot root extracts. Monovalent cations stimulated carrot phosphofructokinase activity. The enzyme was strongly inhibited by P-enolpyruvate and this inhibition was relieved by NACl or KCl. Pi inhibited the enzyme at pH 7.9 but was stimulatory at pH 6.6.     

Localization and capacity of proton pumps in roots of intact sunflower plants

Proton extrusion by roots of intact sunflower plants (Helianthus annuus L.) was studied in nutrient solutions or in agar media with a pH indicator. Proton extrusion was enhanced by either iron deficiency, addition of fusicoccin, or single salt solutions of ammonium or potassium salts. The three types of proton extrusion differ in both localization along the roots and capacity. From their sensitivity to ATPase inhibitors it seems justified to characterize them as proton pumps driven by plasma membrane APTases.

Enhanced proton extrusion induced by preferential cation uptake from (NH₄)₂SO₄ or K₂SO₄ was uniformly distributed over the whole root system. In contrast, the enhancement effect of fusicoccin was confined to the basal root zones and that of iron deficiency to the apical root zones. Also the rates of proton extrusion per unit of root fresh weight differed remarkably and increased in the order: Fusicoccin K₂SO₄ < (NH₄)₂SO₄ < iron deficiency.

Under iron deficiency the average values of proton extrusion for the whole root system are 5.6 micromoles H⁺ per gram fresh weight per hour; however, for the apical root zones values of about 28 micromoles H⁺ can be calculated. This high capacity is most probably related to the iron deficiency-induced formation of rhizodermal transfer cells in the apical root zones. It can be assumed that the various types of root-induced acidification of the rhizosphere are of considerable ecological importance for the plant-soil relationships in general and for mobilization of mineral nutrients from sparingly soluble sources in particular.

     

Localization and compartmentation of Al in the leaves and roots of tea plants

Under acid soil conditions, solubility of aluminum (Al) increases leading to toxicity for plants. Al accumulator species such as tea, however, accumulate high levels of Al in tissues without toxicity symptoms. In this work, Al localization and compartmentation were studied in tea [Camellia sinensis (L.) O. Kuntze] grown hydroponically at 0 or 100 µM Al for eight weeks. Plant dry matter production was significantly higher in the presence of Al and accumulated up to 1.21 and 6.18 mg Al/g DW in the leaves and roots, respectively. About 40-50% of Al was partitioned into cell wall (CW)-bound fraction without any difference among leaves of different age and roots. A significant increase of the soluble phenolics fraction by Al was observed in both leaves and roots. Conventional and confocal laser scanning microscopy images of morin-stained roots indicated a high fluorescence signal in the caps and adjacent mersitematic cells. Towards basal parts, however, Al tended to accumulate mainly in the root hairs, rhizodermal and endodermal cell layers and slightly in the cortex while it was clearly excluded from the central cylinder. A high Al-morin signal was detected from the CW compared with other parts of the cells. Relatively high green fluorescence signal was emitted from the epidermal cell layer, trichomes, vascular bundle region and stomatal cells of particularly young leaves. Our study provides evidences for involvement of both avoidance and tolerance mechanisms for Al in tea plants.     

Effect of apex excision and replacement by 1-naphthylacetic acid on cytokinin concentration and apical dominance in pea plants

As known from literature lateral buds from pea ( Pisum sativum ) plants are released from apical dominance when repeatedly treated with exogenous cytokinins. Little is known, however, about the endogenous role of cytokinins in this process and whether they interact with basipolar transported IAA, generally regarded as the main signal controlling apical dominance. This paper presents evidence that such an interaction exists.
The excision of the apex of pea plants resulted in the release of inhibited lateral buds from apical dominance (AD). This could be entirely prevented by applying 1-naphthylacetic acid (NAA) to the cut end of the shoot. Removal of the apex also resulted in a rapid and rather large increase in the endogenous concentrations of zeatin riboside (ZR), isopentenyladenosine (iAdo) and an as yet unidentified polar zeatin derivative in the node and internode below the point of decapitation. This accumulation of ZR and iAdo, was strongly reduced by the application of NAA. The observed increase in cytokinin concentration preceded the elongation of the lateral buds, suggesting that endogenous cytokinins play a significant role in the release of lateral buds from AD. However, the effect of NAA on the concentration of cytokinins clearly demonstrated the dominant role of the polar basipetally transported auxin in AD. The results suggest a mutual interaction between the basipolar IAA transport system and cytokinins obviously produced in the roots and transported via the xylem into the stem of the pea plants.     

Asymmetric cytokinin signaling opposes gravitropism in roots

Plants depend on gravity to provide the constant landmark for downward root growth and upward shoot growth. The phytohormone auxin and its cell‐to‐cell transport machinery are central determinants ensuring gravitropic growth. Statolith sedimentation toward gravity is sensed in specialized cells. This positional cue is translated into the polar distribution of PIN auxin efflux carriers at the plasma membrane, leading to asymmetric auxin distribution and consequently, differential growth and organ bending. While we have started to understand the general principles of how primary organs execute gravitropism, we currently lack basic understanding of how lateral plant organs can defy gravitropic responses. Here we briefly review the establishment of the oblique gravitropic set point angle in lateral roots and particularly discuss the emerging role of asymmetric cytokinin signaling as a central anti‐gravitropic signal. Differential cytokinin signaling is co‐opted in gravitropic lateral and hydrotropic primary roots to counterbalance gravitropic root growth.     

Antioxidative responses and proline level in leaves and roots of pea plants subjected to nickel stress

The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione S-transferase (GST) as well as proline content were studied in leaves and roots of 14 day-old pea plants treated with NiSO₄ (10, 100, 200 μm) for 1, 3, 6 and 9 days. Exposure of pea plants to nickel (Ni) resulted in the decrease in CuZnSOD as well as total SOD activities in both leaves and roots. The activity of APX in leaves of plants treated with 100 and 200 μm Ni increased following the 3rd day after metal application, while in roots at the end of the experiment the activity of this enzyme was significantly reduced. In both organs CAT activity generally did not change in response to Ni treatment. The activity of GST in plants exposed to high concentrations of Ni increased, more markedly in roots. In both leaves and roots after Ni application accumulation of free proline was observed, but in the case of leaves concentration of this amino acid increased earlier and to a greater extent than in roots. The results indicate that stimulation of GST activity and accumulation of proline in the tissues rather than antioxidative enzymes are involved in response of pea plants to Ni stress.     

Tissue-specific accumulation of carotenoids in carrot roots

Raman spectroscopy can be used for sensitive detection of carotenoids in living tissue and Raman mapping provides further information about their spatial distribution in the measured plant sample. In this work, the relative content and distribution of the main carrot (Daucus carota L.) root carotenoids, α-, β-carotene, lutein and lycopene were assessed using near-infrared Fourier transform Raman spectroscopy. The pigments were measured simultaneously in situ in root sections without any preliminary sample preparation. The Raman spectra obtained from carrots of different origin and root colour had intensive bands of carotenoids that could be assigned to β-carotene (1,520 cm⁻¹), lycopene (1,510 cm⁻¹) and α-carotene/lutein (1,527 cm⁻¹). The Raman mapping technique revealed detailed information regarding the relative content and distribution of these carotenoids. The level of β-carotene was heterogeneous across root sections of orange, yellow, red and purple roots, and in the secondary phloem increased gradually from periderm towards the core, but declined fast in cells close to the vascular cambium. α-carotene/lutein were deposited in younger cells with a higher rate than β-carotene while lycopene in red carrots accumulated throughout the whole secondary phloem at the same level. The results indicate developmental regulation of carotenoid genes in carrot root and that Raman spectroscopy can supply essential information on carotenogenesis useful for molecular investigations on gene expression and regulation.     

Cytokinins in seedling roots of pea

The natural occurrence of cytokinins existing both in a free form and as a constituent of transfer RNA was examined in serial segments of young seedling roots of pea. Purified ethanol extracts of root apices were resolved into four factors capable of inducing soybean callus tissue proliferation. The most active factor was identified as zeatin or some closely related compound; it produced polyploid divisions and tracheary element differentiation when tested on cultured pea root segments. The terminal 0- to 1-millimeter root tip contained 43 to 44 times more free cytokinin on a fresh weight or a per cell basis than the next 1- to 5-millimeter root segment. Extracts of more proximal segments behind the tip contained no measurable free cytokinin. Acid hydrolysates of transfer RNA exhibited reproducible cytokinin activity. Bioassays revealed that the predominant amounts of free cytokinin and that present in transfer RNA were restricted to the extreme root tip. There was approximately 27 times more free cytokinin than the amount detected in transfer RNA in root apices.     

Requirement for chemotaxis in pathogenicity of Agrobacterium tumefaciens on roots of soil-grown pea plants.

Agrobacterium tumefaciens Tn5 mutants deficient in chemotaxis to root exudates were used to study the significance of chemotaxis in crown gall pathogenesis. Mutants deficient in motility and in chemotaxis were fully virulent when inoculated by direct immersion in inoculum, followed by growth for 2 weeks in moist growth pouches. Ability of mutant bacteria to move through soil to infect roots was tested by planting wounded seedlings into air-dried soil or sand that had been infested with inoculum. Mutant bacteria were almost as virulent as the parent on plants grown in sand but were avirulent on soil-grown plants.     

Role and function of cytokinin oxidase in plants

Cytokinin oxidase (CK oxidase) is widely distributed in plants and is the only enzyme that has been shown unequivocally to catalyze the catabolism of specific cytokinins (CKs) to inactive products that lack the N⁶-unsaturated side chain. Thus, the enzyme is thought to play a major role in controlling the level or species of CKs in plant tissues. However, despite its discovery more than 25 years ago, little attention has been given to the elucidation of its role and function in plant growth and development. This review seeks to bring in to context the current state of knowledge regarding the biochemical and molecular properties, regulation in undifferentiated and differentiated tissues, and recent results from studies using transgenic plants in an attempt to provide a more comprehensive understanding of the physiological significance of the enzyme in plants. Notwithstanding species, tissue and other specific differences, in general, CK oxidase appears to contribute to CK homeostasis in plants. However, complete clarity as to its function awaits purification of the protein to homogeneity and the ultimate development of requisite molecular probes.     

Localization of cytokinin responses in the mossFunaria hygrometrica

In the protonema ofFunaria hygrometrica, predetermined cells run through a period of sensitivity to cytokinins, as part of the morphogenetic system with respect to bud formation. The cytokinin sensitivity is highest immediately after the formation of cells in question. The cells can react if enough hormone is present during the sensitive phase. Kinetin is cleaved very rapidly and at an increasing rate. Most probably the concerned enzyme (“kinetin oxidase”) is induced by kinetin. Therefore, cells formed after the beginning of the treatment do not respond by forming buds.     

Regulation of lysine and threonine synthesis in carrot cell suspension cultures and whole carrot roots

Aspartokinase (EC 2.7.2.4), homoserine-dehydrogenase (EC 1.1.1.3) and dihydrodipicolinic-acid-synthase (EC 4.2.1.52) activities were examined in extracts from 1-year-old and 11-year-old cell suspension cultures and whole roots of garden carrot (Daucus carota L.). Aspartokinase activity from suspension cultures was inhibited 85% by 10 mM L-lysine and 15% by 10mM L-threonine. In contrast, aspartokinase activity from whole roots was inhibited 45% by 10 mM lysine and 55% by 10 mM threonine. This difference may be based upon alterations in the ratios of the two forms (lysine-and threonine-sensitive) of aspartokinase, since the activity is consistently inhibited 100% by lysine+threonine. Only one form each of homoserine dehydrogenase and of dihydrodipicolinic acid synthase was found in extracts from cell suspension cultures and whole roots. The regulatory properties of either enzyme were identical from the two sources. In both the direction of homoserine formation and aspartic--semialdehyde formation, homoserine dehydrogenase activities were inhibited by 10mM threonine and 10 mM L-cysteine in the presence of NADH or NADPH. KCl increased homoserine dehydrogenase activity to 185% of control values and increased the inhibitory effect of threonine. Dihydrodipicolinic acid synthase activities from both sources were inhibited over 80% by 0.5 mM lysine. Aspartokinase was less sensitive to inhibition by low concentrations of lysine and threonine than were dihydrodipicolinic acid synthase and homoserine dehydrogenase to inhibition by the respective inhibitors.     

Nitrate-regulated growth and cytokinin responses in seminal roots of barley

The influence of nitrate availability on growth of seminal roots, and root cytokinin levels, was studied in barley (Hordeum vulgare L. cv Golf). Nitrate was continuously supplied to initially N-starved seedlings at relative addition rates (RA) of 0.03 to 0.21 per day (standard cultures) or at RA 0.09 per day in split root cultures with the nitrate additions distributed in ratios of 100:0 or 80:20 to the two subroots. Data were collected both during a phase of acclimation (first 10 days of N additions) and in the acclimated stage (>10 days after onset of N additions). Limitation of whole-plant growth was observed at RA <0.15 per day. The lateral root frequency increased with RA in plants of equal chronological age. However, the lateral root frequency was related to root size rather than to RA; roots of uneven age but having comparable total root lengths also had comparable lateral root frequencies. Growth of individual subroots in split root systems during acclimation was proportional to the fraction of the total N addition that was fed to the root. All subroots had comparable relative growth rates in acclimated plants, and their lateral root frequency correlated with total root length in the same manner as in standard cultures. Onset of N additions in a 80:20 split root culture resulted in doublings of zeatin riboside (ZR) levels in shoots and in the “80” root, whereas the response of the “20” root was small. No effect of perturbed nitrate availability on xylem translocation of ZR was observed. The ZR levels remained higher in the “80” root during acclimation but returned to the level of the “20” root after acclimation. Root cytokinin levels and xylem translocation in acclimated standard cultures were unaffected by RA in the lower range but increased at high RA. Arguments for involvement of cytokinins in the nitrate-regulated growth response are discussed.     

Feedback regulation of xylem cytokinin content is conserved in pea and Arabidopsis

Increased-branching mutants of garden pea (Pisum sativum; ramosus [rms]) and Arabidopsis (Arabidopsis thaliana; more axillary branches) were used to investigate control of cytokinin export from roots in relation to shoot branching. In particular, we tested the hypothesis that regulation of xylem sap cytokinin is dependent on a long-distance feedback signal moving from shoot to root. With the exception of rms2, branching mutants from both species had greatly reduced amounts of the major cytokinins zeatin riboside, zeatin, and isopentenyl adenosine in xylem sap compared with wild-type plants. Reciprocally grafted mutant and wild-type Arabidopsis plants gave similar results to those observed previously in pea, with xylem sap cytokinin down-regulated in all graft combinations possessing branched shoots, regardless of root genotype. This long-distance feedback mechanism thus appears to be conserved between pea and Arabidopsis. Experiments with grafted pea plants bearing two shoots of the same or different genotype revealed that regulation of root cytokinin export is probably mediated by an inhibitory signal. Moreover, the signaling mechanism appears independent of the number of growing axillary shoots because a suppressed axillary meristem mutation that prevents axillary meristem development at most nodes did not abolish long-distance regulation of root cytokinin export in rms4 plants. Based on double mutant and grafting experiments, we conclude that RMS2 is essential for long-distance feedback regulation of cytokinin export from roots. Finally, the startling disconnection between cytokinin content of xylem sap and shoot tissues of various rms mutants indicates that shoots possess powerful homeostatic mechanisms for regulation of cytokinin levels.