Metabolic response in roots of Prunus rootstocks submitted to iron chlorosis

Iron deficiency induces several responses to iron shortage in plants. Metabolic changes occur to sustain the increased iron uptake capacity of Fe-deficient plants. We evaluated the metabolic changes of three Prunus rootstocks submitted to iron chlorosis and their different responses for tolerance using measurements of metabolites and enzymatic activities. The more tolerant rootstocks Adesoto (Prunus insititia) and GF 677 (Prunus amygdalus × Prunus persica), and the more sensitive Barrier (P. persica × Prunus davidiana) were grown hydroponically in iron-sufficient and -deficient conditions over two weeks. Sugar, organic and amino acid concentrations of root tips were determined after two weeks of iron shortage by proton nuclear magnetic resonance spectroscopy of extracts. Complementary analyses of organic acids were performed by liquid chromatography coupled to mass spectrometry. The major soluble sugars found were glucose and sucrose. The major organic acids were malic and citric acids, and the major amino acid was asparagine. Iron deficiency increased root sucrose, total organic and amino acid concentrations and phosphoenolpyruvate carboxylase activity. After two weeks of iron deficiency, the malic, citric and succinic acid concentrations increased in the three rootstocks, although no significant differences were found among genotypes with different tolerance to iron chlorosis. The tolerant rootstock Adesoto showed higher total organic and amino acid concentrations. In contrast, the susceptible rootstock Barrier showed lower total amino acid concentration and phosphoenolpyruvate carboxylase activity values. These results suggest that the induction of this enzyme activity under iron deficiency, as previously shown in herbaceous plants, indicates the tolerance level of rootstocks to iron chlorosis. The analysis of other metabolic parameters, such as organic and amino acid concentrations, provides complementary information for selection of genotypes tolerant to iron chlorosis.     

Characterization of the tolerance to iron chlorosis in different peach rootstocks grown in nutrient solution

Iron chlorosis is an important problem in peach trees, but differences exist between peach rootstocks in their tolerance to Fe chlorosis in calcareous soils. The purpose of this investigation was to characterize the tolerance of different rootstocks to Fe chlorosis induced by bicarbonate in nutrient solution. The rootstocks studied included peach (Nemaguard), plums (Brompton, San Julian A and Puebla de Soto 101) and almond × peach hybrids (Adafuel and GF677). Young plants obtained from rooted cuttings or from in vitro culture techniques were grown individually, under controlled conditions, in flasks with 700 mL of aerated nutrient solution low in iron and with or without 10 mM bicarbonate or 10 mM phosphate. Susceptiblity to bicarbonate-induced chlorosis was inversely correlated with both the Fe content in young leaves and the reducing capacity of roots, but not with the phosphorus content in young leaves. The plum Puebla de Soto 101 and the hybrid GF677 showed the lowest degree of chlorosis and the highest reducing capacity. Phosphate did not induce chlorosis.     

Mapping genetic loci for tolerance to lime-induced iron deficiency chlorosis in grapevine rootstocks (Vitis sp.)

Iron is essential to plants for chlorophyll formation as well as for the functioning of various iron-containing enzymes. Iron deficiency chlorosis is a wide-spread disorder of plants, in particular, of those growing on calcareous soils. Among the different ways to control iron deficiency problems for crops, plant material and especially rootstock breeding is a suitable and reliable method, especially for fruit trees and grapes. The aim of the experiment was to characterize the genetic basis of grapevine chlorosis tolerance under lime stress conditions. A segregating population of 138 F1 genotypes issued from an inter-specific cross between Vitis vinifera Cabernet Sauvignon (tolerant) × V. riparia Gloire de Montpellier (sensitive) was developed and phenotyped both as cuttings and as rootstock grafted with Cabernet Sauvignon scions in pots containing non-chlorosing and chlorosing soils. Tolerance was evaluated by chlorosis score, leaf chlorophyll content and growth parameters of the shoots and roots. The experiments were performed in 2001, 2003 and 2006. The plants analysed in 2006 were reassessed in 2007. The most significant findings of the trial were: (a) the soil properties strongly affect plant development, (b) there are differences in tolerance among segregating genotypes when grown as cuttings or as rootstocks on calcareous soil, (c) calcareous conditions induced chlorosis and revealed quantitative trait loci (QTLs) implicated in polygenic control of tolerance, (d) rootstock strongly contributes to lime-induced chlorosis response, and (e) a QTL with strong effect (from 10 to 25 % of the chlorotic symptom variance) was identified on chromosome 13. This QTL colocalized with a QTL for chlorophyll content (R ² = 22 %) and a major QTL for plant development that explains about 50 % of both aerial and root system biomass variation. These findings were supported by stable results among the different years of experiment. These results open new insights into the genetic control of chlorosis tolerance and could aid the development of iron chlorosis-tolerant rootstocks.     

Genetics of tolerance to iron chlorosis in rice

Genetics of tolerance to iron chlorosis was investigated in eight crosses involving parents distinctly different in their level of tolerance. The segregating populations with parents and F₁s were screened under actual stress conditions in the field. Also, selected crosses were studied for Fe³⁺ uptake capacity. Tolerance/moderate tolerance to Fe chlorosis was dominant over susceptibility and it was controlled by two sets of nonallelic genes with complementary interaction. Gene Ic ₁ has been found to be basic and in complementation with Ic ₃ it confers tolerance. Likewise, Ic ₂ with Ic ₄ confers tolerance. The basic genes Ic ₁ and Ic ₂ are nonallelic and, in the absence of their respective complementary genes Ic ₃ and ₄ , ineffective, which results in susceptibility. Of tolerant cultivars, ARC 10372 and Cauvery have been tentatively assigned the genotype of Ic ₁ , Ic ₂ , Ic ₃ , Ic ₄ , and moderately tolerant IET 7613, Prasanna and Akashi Ic ₁ , ₂ Ic ₃ Ic ₄ . The susceptible ARC 5723 has been assigned Ic ₁ , ₂ , Ic ₃ , Ic ₄ , and IET 9829, Ic ₁ , ₂ Ic ₃ Ic ₄ . IET 7614 is susceptible, due to the presence of inhibitory genes I-Ic ₁ , I-Ic ₂ together with ic ₁ pt>, ic ₂ , Ic ₃ , Ic ₄ . Further, the gene Pc for purple coleoptile shows linkage with one of the complementary genes with a crossover value of 15.26%, while the gene(s) for seedling height Ts with Ic ₁ with a crossover value of 1.7%. It is possible that the gene(s) for iron chlorosis tolerance might belong to the second linkage group, where genes for purple leaf were located.     

Selection of olive varieties for tolerance to iron chlorosis

Under certain conditions, olive trees grown on calcareous soils suffer from iron chlorosis. In the present study several olive varieties and scion-rootstock combinations were evaluated for their tolerance to iron chlorosis. Plants were grown over several months in pots with a calcareous soil, under two fertilization treatments. These consisted of periodic applications of nutrient solutions containing either, 30 μmol/L FeEDDHA or not Fe. Tolerance was assessed by the chlorosis and growth parameters of plants grown without Fe, compared to those plants grown with Fe. Results show that there are differences in tolerance among olive varieties and that tolerance is mainly determined by the genotype of the rootstock. These results open the way to use tolerant varieties for those conditions where iron chlorosis could become a problem.     

Physiological responses and differential gene expression in Prunus rootstocks under iron deficiency conditions

Two Prunus rootstocks, the Myrobalan plum P 2175 and the interspecific peach-almond hybrid, Felinem, were studied to characterize their biochemical and molecular responses induced under iron-Deficient conditions. Plants of both genotypes were submitted to different treatments using a hydroponic system that permitted removal of Fe from the nutrient solution. Control plants were grown in 90 μM Fe (III)-EDTA, Deficient plants were grown in an iron free solution, and plants submitted to an Inductor treatment were resupplied with 180 μM Fe (III)-EDTA over 1 and 2 days after a period of 4 or 15 days of growth on an iron-free solution. Felinem increased the activity of the iron chelate reductase (FC-R) in the Inductor treatment after 4 days of iron deprivation. In contrast, P 2175 did not show any response after at least 15 days without iron. The induction of the FC-R activity in this genotype was coincident in time with the medium acidification. These results suggest two different mechanisms of iron chlorosis tolerance in both Strategy I genotypes. Felinem would use the iron reduction as the main mechanism to capture the iron from the soil, and in P 2175, the mechanism of response would be slower and start with the acidification of the medium synchronized with the gradual loss of chlorophyll in leaves. To better understand the control of these responses at the molecular level, the differential expression of PFRO2, PIRT1 and PAHA2 genes involved in the reductase activity, the iron transport in roots, and the proton release, respectively, were analyzed. The expression of these genes, estimated by quantitative real-time PCR, was different between genotypes and among treatments. The results were in agreement with the physiological responses observed.     

Nitric oxide content is associated with tolerance to bicarbonate-induced chlorosis in micropropagated Prunus explants

Iron (Fe) chlorosis is a common nutritional deficiency in fruit trees grown in calcareous soils. Grafting on tolerant rootstocks is the most efficient practice to cope with it. In the present work, three Prunus hybrid genotypes, commonly used as peach rootstocks, and one peach cultivar were cultivated with bicarbonate in the growth medium. Parameters describing oxidative stress and the metabolism of reactive nitrogen species were studied. Lower contents of nitric oxide and a decreased nitrosoglutathione reductase activity were found in the most sensitive genotypes, characterized by higher oxidative stress and reduced antioxidant defense. In the peach cultivar, which behaved as a tolerant genotype, a specifically nitrated polypeptide was found.     

Storage of in vitro cultures of Prunus rootstocks

In vitro cultures of three Prunus clones (d. 1869, GF 677 and CAB 11E) were successfully stored at +8°, +4° and-3°C following the proliferation phase.Survival of cultures was dependent upon interactions of storage temperature, light, and age of subculture. Up to 100% of the cultures survived at the end of the trials after 170 (at +4°C) and 200 (at-3°C) days storage. Complete dardness appeared more suitable than 16-h (hour) photoperiod for successful storage at-3°C for up to 10 months. One or two weeks in normal growth room vefore storage at-3°C for up to 10 months. One or two weeks in normal growth room before storage enhanced the survival S^-1. The proliferation of the cultures following storage at-3°C in the first subculture appeared similar to those under standard growth room conditions.Part of the results were presented as a poster at the 10th Congress of Eucapia in Wegeningen, The Netherlands, 19–24 June 1983.This paper in No. 504 of the Istituto Coltivazioni Arboree and No. 232 of the Centro Studi Tecnica Frutticola. The research was partially supported by National Research Council (Roma), G.L. Difesa risorse genetiche delle specie arboree.     

A new technique of plant analysis to resolve iron chlorosis

Summary Iron though indispensable for the biosynthesis of chlorophyll, its total content in the plant was not associated with the occurrence of chlorosis. In order to overcome this inconsistency a new technique of plant iron analysis has been developed. It consists of the determination of Fe²⁺, the fraction of iron involved in the synthesis of chlorophyll.The choice of 1–10 o-phenanthroline (o-Ph) as an extractant for Fe²⁺ was based on its remarkably higher stability constant for Fe²⁺ than Fe³⁺. On this basis, it could preferentially chelate Fe²⁺. The highly specific organce colour of the Fe²⁺-phenanthroline complex made possible the determination of Fe²⁺ by reading the transmittancy at 510 nm.The procedure involves extraction of 2 g of thoroughly washed, chopped, fresh plant by 20 ml of o-phenanthroline extractant (pH 3.0, conc. 1.5%). The plant samples treated with the extractant are allowed to stand for 16 hours and Fe²⁺ is determined in the filtrate by reading the transmittancy at 510 nm.In sharp contrast to total iron the green plants always contained more Fe²⁺ than chlorotic plants. The technique has been developed for rice but is expected to be successful for other crops also.     

水稻苗期低温失绿的遗传分析及基因定位

在早季低温条件下, 籼稻品种Dular的幼苗表现出白化失绿, 而粳稻品种Lemont幼苗表现正常绿色。以Lemont和Dular作亲本构建一个F2群体,通过该群体在早季低温条件下性状的表现,发现Lemont和Dular苗期耐冷性的差异受单个主基因控制,低温下白化失绿等位基因为隐性。将该基因暂时命名为cisc(t)。利用该F2群体,采用集团分离分析(BSA)法将cisc(t)定位在9号染色体上。经过对F2群体中100个典型的白化单株的简单序列长度多态性分析,将该基因定位在5.5 cM的区间内,分别与微卫星标记RM257和RM242相距3.9 cM和1.6 cM。     

黄瓜砧用白籽南瓜对不同盐胁迫的耐性评价

采用营养液栽培,研究Ca(NO3)2和NaCl胁迫对黄瓜嫁接用砧木南瓜幼苗生长和抗氧化酶活性的影响,并用隶属函数法综合评价其耐盐性.结果表明:低浓度盐30 mmol·L-1Ca(NO3)2和等渗的45 mmol·L-1 NaCl处理促进砧木幼苗生长;高浓度盐60、120 mmol·L-1Ca(NO3)2和等渗的90、180 mmol·L-1NaCl胁迫下,各砧木幼苗的生长和抗氧化酶系统均受到不同程度的抑制,其中,‘青砧1号’的盐害指数最小,生物量及超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性的下降幅度以及相对电导率的上升幅度均小于其他砧木.高盐Ca(NO3)2胁迫下,各砧木SOD、POD和CAT酶活性均高于等渗的NaCl,而盐害指数和相对电导率低于NaCl,表明Ca(NO3)2对砧木南瓜幼苗生长的危害小于NaCl.4个砧木品种的耐盐性顺序为‘青砧1号’＞‘佐木南瓜’＞‘丰源铁甲’＞‘超霸南瓜’.     

Floral analysis as a tool to diagnose iron chlorosis in orange trees

A three-year field experiment was conducted in a commercial orange grove [Citrus sinensis (L.) Osb. cv. `Valencia late' grafted on Citrange Troyer] established on a calcareous soil in the south of Portugal, to investigate if flower analysis could be used to diagnose lime-induced iron chlorosis. In April, during full bloom, flowers and leaves were collected from 20 trees. Leaf samples were again collected from the same trees in May, June, July and August. Total chlorophyll was estimated in all the leaves sampled for foliar analysis, using a SPAD-502 apparatus. Leaves and flowers were analysed for N, P, K, Ca, Mg, Fe, Zn, Mn and Cu. Principal Component Analysis was used to evaluate the variation of nutrient concentrations in flowers, and linear regressions were established between these and the chlorophyll content of leaves 90 days after full bloom. Evaluation of the best-fit equation was carried out using separate data obtained from other groves. Variation in the pattern of floral mineral composition in the flowers showed contrasts between the increase in N, P and K and that of Ca, Fe and Zn, while the concentration of Mg, Mn and Ca varied synchronously. The ratio of Mg:Zn in flowers explained about half of the variation of chlorophyll in leaves later in the season. A ratio below 100 indicated that trees would develop iron chlorosis, while with a ratio above 200 leaves would remain green. An early prognosis of iron chlorosis based on floral analysis can benefit growers, since it allows them to apply treatments in time to prevent loss of fruit yield and quality due to iron chlorosis.     

Responses of two Prunus rootstocks to KCl induced salinity in vitro

The in vitro response of two Prunus rootstocks: GF 677 (Prunus persica × Prunus amygdalus), and Nemared (Prunus persica) to increasing concentrations of KCl of the culture medium was studied. Shoots were grown in vitro for 8 weeks on an Murashige and Skoog medium supplemented with 0, 5, 10, 15, 20, 40 or 80 mM KCl. By increasing KCl concentration from 0 to 40 mM, the number of shoots per explant was not significantly affected for both rootstocks. However, Nemared rootstock formed more shoots per explant than GF 677 under respective KCl concentrations of the medium. Inclusion of 80 mM KCl in the medium resulted in a reduction of growth of both rootstocks. Sodium, Fe, Mn, and Zn concentration in tissues of Nemared rootstock were significantly higher than the respective values of GF 677.     

Effect of iron chlorosis on mineral nutrition and lipid composition of thylakoid biomembrane in Prunus persica (L.) Bastch.

The effect of iron chlorosis on mineral, thylakoid lipids and fatty acids composition of field grown peach tree leaves was studied. Significant differences were found in iron extracted by using , -dipyridyl (active iron), total iron, P, K, Cu and the P/Fe and Fe/Mn ratios. The levels of total chlorophyll, total glycolipids and phospholipids were reduced under iron chlorosis. A slight iron deficiency does not modify the fatty acid composition of thylakoid membranes, while a strong deficiency changes the proportion of some fatty acids.Abbreviations Chl chlorophyll - DGDG digalactosyldiglycerol - MGDG mono-galactosyldiglycerol - PC phosphatidycholine - PE phophatidylethanolamine - PG phophatidylglycerol - TLC thin layer chromatography - 16:0 palmitic acid - 16:1 palmitoleic acid - 16:lt trans-hexadecenoic - 18:0 steric acid - 18:1 oleic acid - 18:2 linoleic acid - 18:3 linolenic acid     

Nitrate induced iron deficiency chlorosis in Juncus acutiflorus

Chlorosis caused by iron deficiency is commonly associated with high bicarbonate levels in the soil. However, in rare cases such chlorosis has been observed in soils with high nitrate levels. In a dutch rich-fen, chlorosis has been noted in stands of Juncus acutiflorus at locations where groundwater containing high levels of nitrate reached the surface. Experiments revealed that the chlorosis could be attributed to iron deficiency although iron levels in the shoots were well above the known physiological threshold values for iron deficiency. It is postulated that increased nitrate assimilation leads to an increased apoplastic pH and to a concomitant immobilisation of iron and/or lower iron (III) reduction. Moreover free amino acid levels were markedly higher in the iron deficient plants in the field. It was found, however, that the percentage of nitrogen present as free amino acids was not influenced directly by low iron levels but mainly by the C/N ratios in the shoots. Nowadays, nitrate concentrations in ground water as high 1000 µM are no longer an exception in the Netherlands. We propose that strongly increased nitrate inputs may cause iron stress in natural vegetations, especially in wet habitats.     

Simple sequence repeat-based assessment of genetic relationships among Prunus rootstocks

Ten SSR loci, previously developed for Prunus, were analyzed to examine genetic relationships among 23 rootstock candidates for sweet and sour cherries, of the species P. avium, P. cerasus, P. mahaleb, and P. angustifolia. Five genotypes of P. laurocerasus, not used as rootstock, were included in the molecular analysis. The number of alleles per locus ranged from 8 to 12, with a mean of 9, while the number of microsatellite genotypes varied from 8 to 17, indicating that the SSRs were highly informative. The degree of heterozygosity (0.61) was high. Clustering analysis resulted in two main clusters. The first cluster was divided into two subclusters; the first subcluster consisted of P. avium and P. cerasus, and the second subcluster consisted of P. laurocerasus. The second cluster was divided into two subclusters. The first subcluster consisted of P. mahaleb genotypes and the second consisted of P. angustifolia genotypes. The reference rootstocks also clustered with their associated botanical species. Unweighted pair-group method with arithmetic mean analysis demonstrated that P. laurocerasus genotypes had less genetic variation and that P. avium genotypes were more closely related to P. cerasus. The SSR-based phylogeny was generally consistent with Prunus taxonomy information, suggesting the applicability of SSR analysis for genotyping and phylogenetic studies in the genus Prunus.     

Genetic analysis of NaCl tolerance in tomato

We attempted to find the suitable parents for the development of tomato hybrids for high salt soils by exploiting combining ability, gene action and heterosis. Six salt-tolerant and three salt-intolerant genotypes, along with their 18 F1 crosses, were evaluated at seedling stage under 10 and 15 dS/m (NaCl) salinity stress, compared to the control level of salinity. The experiment was laid out based on a two-way complete randomized design factorial arrangement with two replications; data on root and shoot length, fresh and dry weights, leaf area, plant length, Na(+), K(+) and K(+)/Na(+) concentrations were recorded. There was significant variation within genotypes, lines, testers, crosses, and line × tester interaction for all plant characters studied under normal and two salinity levels. Estimates of combining ability indicated that under low (10 dS/m) and high (15 dS/m) salinities, line BL1176 and tester LO2875 showed significant GCA effects for most of the traits studied. The cross-combinations 6233 × LO2875, CLN2498A x LO2875 and BL1176 × 17902 showed highest SCA values for most of the characters under 10 and 15 dS/m, respectively. Potence ratio showed that under low and high salinities, all the traits showed over dominant type of gene action except leaf area and K(+) concentration (in 10 dS/m) and shoot length, and leaf area (in 15 dS/m). The highest heterosis for most of the parameters was observed in cross-combinations BL1176 × LO2875 and CLN2498A x LO2875.     

Varietal response to iron chlorosis in upland rice

Summary In a study involving twenty upland rice genotypes, the induction of iron chlorosis was found to be more by superphosphate application than due to lime incorporation into an alfisol soil under greenhouse conditions in GI tray-grown seedlings as quantified by two parameters viz., total chlorphyll content and Fe²⁺ content. Of the two indices of iron chlorosis, Fe²⁺ was more sensitive than chlorophyll content. Genotypes were grouped into efficient and inefficient categories both in terms of absorption and utilization of iron based on the degree of reduction in response to added superphosphate.     

Effect of meta-Topolin on micropropagation and adventitious shoot regeneration in Prunus rootstocks

The effect of meta-Topolin (mT), an aromatic natural cytokinin, on micropropagation and adventitious shoot regeneration was evaluated on Prunus rootstocks, Torinel (Prunus domestica L.) and Ferdor (Prunus insititia  ×  domestica). In vitro shoots were grown for three subcultures on a multiplication medium containing 2.1, 4.2 or 6.3 μM of mT or 2.1 µM N ⁶-benzyladenine (BA). Then, apical leaves were excised and transferred on a medium supplied with BA, thidiazuron (TDZ) or zeatin for adventitious regeneration. Shoots multiplied on 2.1 μM mT or BA, were also induced to root with α-naphthalene acetic acid and acclimatized. meta-Topolin did not improve shoot proliferation, respect to BA, however, positively influences growth and quality of shoots. Ferdor from mT showed higher rooting percentage (92 %), root number and length, respect to the control, while a similar response was observed in Torinel with both cytokinins. Acclimatisation was higher than 90 % for both genotypes and, after 5 months, the highest length of roots was found in plants from mT. Adventitious regeneration was obtained only in leaves from shoots previously grown on mT. The highest regeneration responses, 65 and 42 %, respectively for Ferdor and Torinel, were obtained in the regeneration medium supplied with TDZ.