Technical advance: reduction of Fe(III)-chelates by mesophyll leaf disks of sugar beet. Multi-component origin and effects of Fe deficiency

The characteristics of the Fe(III)-chelate reductase activity have been investigated in mesophyll disks of Fe-sufficient and Fe-deficient sugar beet leaves. The Fe(III)-chelate reductase activity of mesophyll disks was light dependent and increased markedly when the epidermis was removed. Iron(III)-citrate was photo-reduced directly by light in the absence of plant tissue. Total reductase activity was the sum of enzymatic mesophyll reduction, enzymatic reduction carried out by organelles exposed at the disk edge and reduction caused by the release of substances both by exposed mesophyll cells and at the disk edge. Compounds excreted were shown by HPLC to include organic anions, mainly oxalate, citrate and malate. When expressed on a leaf surface basis, Fe deficiency decreased the total mesophyll Fe(III)-chelate reductase activity. However, Fe-sufficient disks reduced less Fe than the Fe-deficient ones when expressed on a chlorophyll basis. The optimal pH values for Fe(III) reduction were always in the range 6.0-6.7. In control leaves Fe(III)-citrate and Fe(III)-malate were the substrates that led to the highest Fe reduction rates. In Fe-deficient leaves Fe(III)-malate led to the highest Fe reduction rates, followed by Fe(III)-EDTA and then Fe(III)-citrate. K:(m) values for the total reductase activity, enzymatic mesophyll reduction and enzymatic reduction carried out by organelles at the disk edge were obtained.     

Effects of cadmium and lead on ferric chelate reductase activities in sugar beet roots

The effects of the heavy metals Cd and Pb on the activity of the enzyme ferric chelate reductase (FC-R, E.C. 1.6.99.13) have been studied in excised sugar beet root tips. The activity of this enzyme is markedly increased by iron deficiency. Metals were used as chloride salts or chelated with EDTA, and chemical speciation was carried out to predict the metal chemical species in equilibrium both in the ferric reductase assay and in the nutrient solutions. Three different heavy metal treatments were used. First, effects of Cd and Pb on the functioning of the FC-R were assessed in Fe-deficient plants, by including metals in the enzyme assay medium only. Results indicate that 50 μM CdCl₂ or Cd-EDTA did not affect FC-R activities even when assay time was as long as 2 h, whereas Pb slightly decreased enzyme activity only at concentrations of 2 mM. Second, short-time Cd and Pb pre-treatments (30–60 min) were imposed on intact Fe-deficient plants before carrying out the assay of FC-R activity. These short-term treatments induced significant decreases in the FC-R activities previously induced by Fe deficiency. With Cd, effects were more pronounced at higher concentrations, and they were stronger when Cd was in the free ion form than when present in the form of Cd-EDTA chelate. Third, prolonged Cd and Pb treatments were imposed on plants grown on 45 μM Fe-EDTA to assess the long-term effects of heavy metals on the induction of the FC-R enzyme. These long-term heavy metal treatments caused a significant increase in the root FC-R activities, indicating that Cd and Pb induce a deficiency in Fe in sugar beet that in turn elicits FC-R activity. The increases, however, are not as large as those found in total absence of Fe.     

Iron deficiency-associated changes in the composition of the leaf apoplastic fluid from field-grown pear (Pyrus communis L.) trees.

Experiments have been carried out with field-grown pear trees to investigate the effect of iron chlorosis on the composition of the leaf apoplast. Iron deficiency was associated with an increase in the leaf apoplastic pH from the control values of 5.5-5.9 to 6.5-6.6, as judged from direct pH measurements in apoplastic fluid obtained by centrifugation and fluorescence of leaves incubated with 5-CF. The major organic acids found in leaf apoplastic fluid of iron-deficient and iron-sufficient pear leaves were malate, citrate and ascorbate. The total concentration of organic acids was 2.9 mM in the controls and increased to 5.5 mM in Fe-deficient leaves. The total apoplastic concentration of inorganic cations (Ca, K and Mg) increased with Fe deficiency from 15 to 20 mM. The total apoplastic concentration of inorganic anions (Cl-, NO3-, SO4(2-) and HPO4(2-)) did not change with Fe deficiency. Iron concentrations decreased from 4 to 1.6 microM with Fe deficiency. The major Fe species predicted to exist in the apoplast was [FeCitOH](-1) in both Fe-sufficient and deficient leaves. Organic acids in whole leaf homogenates increased from 20 to 40 nmol x m(-2) with Fe deficiency. The accumulation of organic anions in the Fe-deficient leaves does not appear to be associated to an increased C fixation in leaves, but rather it seems to be a consequence of C transport via xylem.     

Fe Resupply to Fe-deficient Sugar Beet Plants Leads to Rapid Changes in the Violaxanthin Cycle and other Photosynthetic Characteristics without Significant de novo Chlorophyll Synthesis

The effects of Fe resupply to Fe-deficient plants have been investigated in hydroponically-grown sugar beet. In the short-term (24 h) after Fe resupply, major changes were observed, although de novo chlorophyll (Chl) synthesis had not begun yet. Approximately 50% of the zeaxanthin was converted into violaxanthin, whereas the actual Photosystem II (PS II) efficiency increased by 69% and non-photochemical quenching (NPQ) and the amount of thermally dissipated energy decreased markedly (by 47% and 40%, respectively). At the same time, photosynthetic rate increased approximately by 50%. From one to two days after Fe resupply, there was a gradual increase in the leaf concentrations of Chl and other photosynthetic pigments, accompanied by a further conversion of zeaxanthin into violaxanthin, increases in actual PS II efficiency and photosynthetic rates and decreases in NPQ and the amount of thermally dissipated energy. At 72-96 h after Fe resupply, leaf pigment concentrations, photosynthetic rates and actual PS II efficiency had increased further, although both photosynthetic rate and leaf pigment concentrations were still lower than those found in Fe-sufficient leaves. Good correlations were observed between the amount of light thermally dissipated by the PS II antenna, NPQ and the antheraxanthin + zeaxanthin concentration after Fe resupply, confirming the photoprotective role of the xanthophyll cycle in Fe-deficient sugar beet leaves. Similar correlations were observed for lutein, suggesting a possible role of this pigment in photoprotection.     

Leaf structural changes associated with iron deficiency chlorosis in field-grown pear and peach: physiological implications

Plants grown in calcareous, high pH soils develop Fe deficiency chlorosis. While the physiological parameters of Fe-deficient leaves have been often investigated, there is a lack of information regarding structural leaf changes associated with such abiotic stress. Iron-sufficient and Fe-deficient pear and peach leaves have been studied, and differences concerning leaf epidermal and internal structure were found. Iron deficiency caused differences in the aspect of the leaf surface, which appeared less smooth in Fe-deficient than in Fe-sufficient leaves. Iron deficiency reduced the amount of soluble cuticular lipids in peach leaves, whereas it reduced the weight of the abaxial cuticle in pear leaves. In both plant species, epidermal cells were enlarged as compared to healthy leaves, whereas the size of guard cells was reduced. In chlorotic leaves, bundle sheaths were enlarged and appeared disorganized, while the mesophyll was more compacted and less porous than in green leaves. In contrast to healthy leaves, chlorotic leaves of both species showed a significant transient opening of stomata after leaf abscission (Iwanoff effect), which can be ascribed to changes found in epidermal and guard cells. Results indicate that Fe-deficiency may alter the barrier properties of the leaf surface, which can significantly affect leaf water relations, solute permeability and pest and disease resistance.     

Effects of iron deficiency on the composition of the leaf apoplastic fluid and xylem sap in sugar beet. Implications for iron and carbon transport

The effects of iron deficiency on the composition of the xylem sap and leaf apoplastic fluid have been characterized in sugar beet (Beta vulgaris Monohil hybrid). pH was estimated from direct measurements in apoplastic fluid and xylem sap obtained by centrifugation and by fluorescence of leaves incubated with 5-carboxyfluorescein and fluorescein isothiocyanate-dextran. Iron deficiency caused a slight decrease in the pH of the leaf apoplast (from 6.3 down to 5.9) and xylem sap (from 6.0 down to 5.7) of sugar beet. Major organic acids found in leaf apoplastic fluid and xylem sap were malate and citrate. Total organic acid concentration in control plants was 4.3 mM in apoplastic fluid and 9.4 mM in xylem sap and increased to 12.2 and 50.4 mM, respectively, in iron-deficient plants. Inorganic cation and anion concentrations also changed with iron deficiency both in apoplastic fluid and xylem sap. Iron decreased with iron deficiency from 5.5 to 2.5 microM in apoplastic fluid and xylem sap. Major predicted iron species in both compartments were [FeCitOH](-1) in the controls and [FeCit(2)](-3) in the iron-deficient plants. Data suggest the existence of an influx of organic acids from the roots to the leaves via xylem, probably associated to an anaplerotic carbon dioxide fixation by roots.     

Changes in sugar beet leaf plasma membrane Fe(III)-chelate reductase activities mediated by Fe-deficiency, assay buffer composition, anaerobiosis and the presence of flavins

Summary Different assay conditions induce changes in the ferric chelate reductase activities of leaf plasma membrane preparations from Fe-deficient and Fe-sufficient sugar beet. With an apoplasttype assay medium the ferric chelate reductase activities did not change significantly when Fe(III)-EDTA was the substrate. However, with ferric citrate as substrate, the effect depended on the citrateto-Fe ratio. When the citrate-to-Fe ratio was 20 1, the effects were practically unappreciable. However, with a lower citrate-to-Fe ratio of 5 1 the activities were significantly lower with the apoplast-type medium than with the standard assay medium. Our data also indicate that anaerobiosis during the assay facilitates the reduction of ferric malate and Fe(III)-EDTA by plasma membrane preparations. Anaerobiosis increased by approximately 50% the plasma membrane ferric chelate reductase activities when Fe(III)-EDTA was the substrate. With ferric malate anaerobiosis increased activities by 70–90% over the values obtained in aerobic conditions. However, with ferric citrate the increase in activity by anaerobiosis was not significant. We have also tested the effect of riboflavin, flavin adenine dinucleotide, and flavin mononucleotide on the plasma membrane ferric chelate reductase activities. The presence of flavins generally increased activities in plasma membrane preparations from control and Fe-deficient plants. Increases in activity were generally moderate (lower than twofold). These increases occurred with Fe(III)-EDTA and Fe(III)-citrate as substrates.Abbreviations BPDS bathophenantroline disulfonate - FC ferric chelate - FC-R ferric chelate reductase - PM plasma membrane     

Stress-dependent redistribution of abscisic acid (ABA) in Hordeum vulgare L leaves: the role of epidermal ABA metabolism, tonoplastic transport and the cuticle

When ¹⁴C-labelled abscisic acid ([¹⁴C]ABA) was supplied to isolated protoplasts of the barley leaf at pH 6, initial rates of metabolism were about five times higher in epidermal cell protoplasts than in mesophyll cell protoplasts if equal cytosolic volumes were considered. In spite of the fact that epidermal cells make up only about 35% of the total water space in barley leaves, and despite the small cytosolic volume of these cells, in intact leaves all epidermal cells would thus metabolize half as much ABA per unit time as the mesophyll cells (0–27 and 0–51 mmol h^?1 m^?3 leaf water). Therefore, under these conditions epidermal cells seem to be a stronger sink than mesophyll cells for ABA that arrives via the transpiration stream. However, at an apoplastic pH of 7–25, which occurs in stressed leaves, the proportion of total metabolized ABA would be much smaller in epidermal than in mesophyll cells (0–029 and 0–204 mmolh^?l m^?3 leaf water). Our results indicate that under conditions of slightly alkaline apoplastic pH the epidermis may serve as the main source for fast stress-dependent ABA redistribution into the guard cell apoplast. This is partly the result of ABA transport across the epidermal tonoplast, which is dependent on the apoplastic pH and possibly on the cytosolic calcium concentration. The cuticle seems to be of no particular importance in stress-induced apoplastic ABA shifts and cannot be regarded as a significant sink for high ABA concentrations under stress.     

Increases in phosphoenolpyruvate carboxylase activity in iron-deficient sugar beet roots: Analysis of spatial localization and post-translational modification

Root tips of Fe-deficient and Fe-sufficient sugar beet plants grown in hydroponics have been used to study the changes in the amount and activity of the cytosolic enzyme phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31). Phosphoenolpyruvate carboxylase activity in extracts of the yellow Fe-deficient root tips was, at pH 7.3, 30-fold higher (when expressed on a FW basis) and 7.1-fold higher (when expressed on a protein basis) than that found in the extracts of Fe-sufficient root tips. The amount of phosphoenolpyruvate carboxylase protein determined by immuno-blotting was, on a protein basis, 35-fold larger in the yellow zone of Fe-deficient root tips than in the Fe-sufficient root tips. The inhibition of the phosphoenolpyruvate carboxylase activity by 500 m malate was 41 and 58% in the extracts Fe-deficient and Fe-sufficient roots. The possibility that post-translational regulation of phosphoenolpyruvate carboxylase may occur mediated through phosphorylation, was studied by immunological detection of phosphoserine residues in root tip extracts.     

Limiting Factors in Photosynthesis: VI. Regeneration of Ribulose 1,5-Bisphosphate Limits Photosynthesis at Low Photochemical Capacity

Earlier work (SE Taylor, N Terry [1984] Plant Physiol 75: 82-86) has shown that the rate of photosynthesis may be colimited by photosynthetic electron transport capacity, even at low intercellular CO₂ concentrations. Here we monitored leaf metabolites diurnally and the activities of key Calvin cycle enzymes in the leaves of three treatment groups of sugar beet (Beta vulgaris L.) plants representing three different in vivo photochemical capacities, i.e. Fe-sufficient (control) plants, moderately Fe-deficient, and severely Fe-deficient plants. The results show that the decrease in photosynthesis with Fe deficiency mediated reduction in photochemical capacity was through a reduction in ribulose 1,5-bisphosphate (RuBP) regeneration and not through a decrease in ribulose 1,5-bisphosphate carboxylase/oxygenase activity. Based on measurements of ATP and NADPH and triose phosphate/3-phosphoglycerate ratios in leaves, there was little evidence that photosynthesis and RuBP regeneration in Fe-deficient leaves were limited directly by the supply of ATP and NADPH. It appeared more likely that photochemical capacity influenced RuBP regeneration through modulation of enzymes in the photosynthetic carbon reduction cycle between fructose-6-phosphate and RuBP; in particular, the initial activity of ribulose-5-phosphate kinase was strongly diminished by Fe deficiency. Starch and sucrose levels changed independently of one another to some extent during the diurnal period (both increasing in the day and decreasing at night) but the average rates of starch or sucrose accumulation over the light period were each proportional to photochemical capacity and photosynthetic rate.     

Contamination of oat mesophyll protoplasts by apoplastic polyamine oxidase

Mesophyll protoplasts isolated from peeled oat ( Avena sativa L. cv Victory) leaves with 1% (w/v) Cellulysin in 20 m M KPO₄, pH 5.5 and 0.6 M sorbitol retain about 6% of the polyamine oxidase (PAO, EC 1.4.3.4) activity of the whole peeled leaf. However, more than 99% of the oat leaf PAO activity is apoplastic and can be extracted by vacuum infiltration with 200 m M NaCl and this procedure extracts no activity for the cytoplasmic marker enzyme glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49). By these criteria we consider PAO in oat leaves to be totally apoplastic and PAO found in the isolated protoplast to be contamination. The degree of protoplast contamination by PAO depends on the pH and ionic strength of the isolating and washing medium. It can be eliminated by washing protoplasts in 0.6 M sorbitol with 100 m M KPO₄, pH 6.5. Pellets of lysed protoplasts incubated with dialyzed apoplastic enzymes in 5 m M KPO₄, pH 5.5 adsorb about 87% of the added PAO activity but only about 25% of the added peroxidase (EC 1.11.1.7) activity. The adsorbed activity can be solubilized from the pellet by extraction with 1 M NaCl. The results demonstrate that weakly ionically bound cell wall enzymes may contaminate protoplasts isolated and purified by conventional techniques.     

Phytosulfokine stimulates cell divisions in sugar beet (Beta vulgaris L.) mesophyll protoplast cultures

The aim of this work was to improve plating efficiency of sugar beet mesophyll protoplast cultures. Preliminary experiments showed that cultures of good quality, viable protoplasts were obtained in rich media based on the Kao and Michayluk formulation and with the calcium alginate as an embedding matrix. Nevertheless, in these cultures cell divisions were either not observed or very seldom confirming earlier reported recalcitrance of sugar beet protoplasts. The recalcitrant status of these cultures was reversed upon application of exogenous phytosulfokine (PSK)—a peptidyl plant growth factor. The highest effectiveness of PSK was observed at 100 nM concentration. Plating efficiencies obtained in the presence of PSK reached approximately 20% of the total cultured cells. The stimulatory effect of phytosulfokine was observed for all tested breeding stocks of sugar beet. Our data indicate that PSK is a powerful agent able to overcome recalcitrance of plant protoplast cultures.     

Uptake of 5 9Fe from soluble 5 9Fe-humate complexes by cucumber and barley plants

The capability of cucumber (Cucumis sativus L., cv. Serpente cinese), a Strategy I plant and barley (Hordeum vulgaris L., cv. Europa), a Strategy II plant to use Fe complexed by a water-soluble humic fraction (WEHS) extracted from a peat, was studied. Uptake of ⁵⁹Fe from ⁵⁹Fe-WEHS by cucumber plants was higher at pH 6.0 than at pH 7.5. Roots of intact cucumber plants were able to reduce the Fe^III-WEHS complex either at pH 6.0 or 7.5, rates being higher in the assay medium buffered at pH 6.0. After supply of ⁵⁹Fe-WEHS, a large pool of root extraplasmatic ⁵⁹Fe was formed, which could be used to a large extent by Fe-deficient plants, particularly under acidic conditions. Uptake of ⁵⁹Fe from ⁵⁹Fe-WEHS by Fe-sufficient and Fe-deficient barley plants was examined during periods of high (morning) and low (evening) PS release. Uptake paralleled the diurnal rhythm of PS release. Furthermore, ⁵⁹Fe uptake was strongly enhanced by addition of PS to the uptake solution in both Fe-sufficient and Fe-deficient plants. High amount of root extraplasmatic ⁵⁹Fe was formed upon supply of Fe-WEHS, particularly in the evening experiment. Fe-deficient barley plants were able to utilize Fe from the root extraplasmatic pool, conceivably as a result of high rates of PS release. The results of the present work together with previous observations indicate that cucumber plants (Strategy I) utilize Fe complexed to WEHS, presumably via reduction of Fe^III-WEHS by the plasma membrane-bound reductase, while barley plants (Strategy II) use an indirect mechanism involving ligand exchange between WEHS and PS.     

几种植物原生质体的扫描电镜观察

扫描电镜观察表明,分离自马铃薯、萱草。甘蔗、木薯和落花生等不同植物和组织的原生质体表面呈现不同程度的凹凸不平。马铃薯叶肉原生质体表面较粗糙,其余四种植物叶肉、幼茎或子叶原生质体稍光滑。有的原生质体显现不同程度的凹陷现象。有的原生质体表面尚残留有未完全水解的胞壁碎片。在木薯幼茎原生质体制备物中见有呈“裂片”状的球形结构。原生质体表面扫描图象的差异似与不同种植物有关,与组织源不同更有密切关系。 原生质体镀膜前,涂布于已镀膜的盖玻片支持物上的原生质体很少或无凹陷现象,涂布于已镀膜的双面胶支持物上的原生质体凹陷严重。     

Improvement of protoplast culture protocols for Beta vulgaris L. (sugar beet)

Summary The effects of NaCl, feeder cells and the embedding of protoplasts in calcium alginate have been investigated in an attempt to improve culture conditions of recalcitrant sugar beet (Beta vulgaris L.) mesophyll protoplasts. While the use of NaCl in all instances proved detrimental to protoplast development, the other two treatments had clear beneficial effects. Minimum plating densities, necessary to sustain cell division, could be reduced to <5% (<4000 protoplasts / ml) of the control levels and plating efficiencies could be significantly enhanced by approx. 10 fold. Plants could still be regenerated from soft calli derived from mesophyll protoplasts cultured under the modified conditions at a frequency of 20–30 %. In particular, the use of alginate is considered of potentially great importance for the further application of beet protoplasts for other aims e.g. asymmetric hybridization.     

小麦原生质体分离过程中生理状态的变化

酶解处理使小麦对肉原生质体膜流动性降低,膜脂过氧化产物丙二醛（ＭＤＡ）积累,说明脱璧过程对细胞有伤害作用,损伤位点可能发生在膜上。胚性愈伤组织的具有分裂能力的原生质体,不表现上述变化。酶解脱壁还使超氧化物歧化酶（ＳＯＤ）和过氧化氢酶（ＣＡＴ）活性上升;过氧化物酶（ＰＯＸ）在叶肉原生质体中活性下降,在胚性愈伤组织来源的原生质体中活性上升。以上结果表明：在原生质体分离过程中,细胞的生理特性发生了变化;膜损伤的发生可能与原生质体能否进入正常分裂状态有关。     

Cell-wall antigens in mesophyll cells and mesophyll-derived protoplasts of sugar beet: possible implication in protoplast recalcitrance?

We have investigated the possible relation between plant cell-wall constituents and the recalcitrance of the cell to regenerate organs and whole plants in vitro. A temporal and spatial expression of several carbohydrate epitopes was observed both within leaf tissue used for protoplast isolation and within new walls reformed by recalcitrant mesophyll protoplasts of sugar beet ( Beta vulgaris L.); these include four pectic epitopes, one xyloglucan (rhamnogalacturonan I) epitope, two carbohydrate motifs of arabinogalactan proteins (AGPs) and callose. The walls of mesophyll cells and newly formed walls of protoplasts were similar with respect to the presence of large amounts of pectins recognized by JIM7 antibodies, the scarcity of JIM5-pectins and the complete absence of LM5-responding pectin molecules. Their main differences were the significantly higher accumulation of LM6-recognizing pectins and the very conspicuous greater accumulation of AGPs and callose in walls deposited by protoplasts than in those synthesized by donor cells.     

水杨酸和茉莉酸甲酯对丹参幼苗叶片显微结构、光合及非结构糖积累的影响

研究了水杨酸(SA)和茉莉酸甲酯(MeJA)处理对丹参(Salvia miltiorrhiza Bunge)幼苗叶片显微结构、叶片光合能力及幼苗中非结构糖积累的影响.结果显示:SA处理增加了丹参幼苗叶片气孔密度;叶肉细胞排列紧密、体积减小,叶肉细胞内叶绿体数目减少,但叶绿体体积增大,叶绿体基粒片层结构的数目增加;叶片中叶绿素a、b含量、叶气孔导度、蒸腾速率以及净光合速率均增加;同时,幼苗根中和叶片中酸性转化酶活性降低,幼苗地上部分蔗糖含量及可溶性糖总量显著高于对照.MeJA处理减少了叶片气孔密度,气孔发育畸形;叶肉细胞间隙增大,栅栏细胞层数减少,叶肉细胞内叶绿体数目减少,叶绿体体积减小,叶绿体基粒片层结构被破坏;叶片中叶绿素a及类胡萝卜素含量、叶片的净光合速率低于对照,叶气孔导度、蒸腾速率增强;同时,幼苗根中及叶中酸性转化酶活性增加,幼苗根中蔗糖含量及可溶性糖总量显著低于对照.可见,SA处理能促进植物叶片显微结构发育,增强叶片光合能力,抑制蔗糖降解并促进蔗糖积累;而MeJA处理则破坏了植物叶片显微结构,降低了叶片光合能力,促进了蔗糖降解并减少蔗糖积累.