Study on Citrus Response to Huanglongbing Highlights a Down-Regulation of Defense-Related Proteins in Lemon Plants Upon ‘Ca. Liberibacter asiaticus’ Infection

Citrus huanglongbing (HLB) is a highly destructive disease of citrus presumably caused by ‘ Candidatus Liberibacter asiaticus ’ (Las), a gram-negative, insect-transmitted, phloem-limited α-proteobacterium. Although almost all citrus plants are susceptible to HLB, reports have shown reduced susceptibility to Las infection in lemon ( Citrus limon ) plants. The aim of this study is to identify intra-species specific molecular mechanisms associated with Las-induced responses in lemon plants. To achieve this, comparative 2-DE and mass spectrometry, in addition to Inductively Coupled Plasma Spectroscopy (ICPS) analyses, were applied to investigate differences in protein accumulation and the concentrations of cationic elements in leaves of healthy and Las-infected lemon plants. Results showed a differential accumulation of 27 proteins, including an increase in accumulation of starch synthase but decrease in the production of photosynthesis-related proteins in Las-infected lemon plants compared to healthy plants. Furthermore, there was a 6% increase (P > 0.05) in K concentration in leaves of lemon plants upon Las infection, which support results from previous studies and might represent a common response pattern of citrus plants to Las infection. Interestingly, contrary to reports from prior studies, this study showed a general reduction in the production of defense-related pathogen-response proteins but a 128% increase in Zn concentration in lemon plants in response to Las infection. Taken together, this study sheds light on general and intra-species specific responses associated with the response of citrus plants to Las.     

The intrinsic zinc atoms of yeast alcohol dehydrogenase

The intrinsic Zn content of yeast alcohol dehydrogenase (YADH) has been determined by three highly sensitive analytical techniques. The enzyme prepared from baker's yeast has a specific activity of 430–460 U/mg and contains 4 intrinsically bound Zn atoms per tetrameric enzyme of molecular weight 150,000. The enzyme is homogeneous by disc gel electrophoresis and analytical ultracentrifugation and remains stable and fully active on prolonged storage. YADH samples from commercial sources, while of high activity, can initially contain more than 4 g-atom of Zn/mole, but dialysis against EDTA removes these adventitious Zn atoms which do not bear a consistent relationship to enzymatic activity, in accord with earlier investigations. Apparently, they are bound to the enzyme in a manner different from that of the catalytically essential Zn atoms and likely represent contamination. The 4 intrinsic Zn atoms exchange fully with ⁶⁵Zn(II) resulting in [(YADH)⁶⁵Zn₄] which exhibits the same specific activity and stability as the native enzyme.     

Effect of Manganese Deficiency on Chloroplasts of Lemon Leaves

Manganese deficiency in chloroplasts of Eureka lemon leaves resulted in 23% and 40% increase of chloroplast nitrogen and protein, respectively, on a chlorophyll unit basis. Acrylamide gel electrophoresis carried out on extracts of these chloroplasts disclosed also qualitative differences between the normal and deficient leaves. Calculated on chloroplast N basis there is an increase of 17% in the chloroplast protein under Mn deficiency. This increase apparently indicates a more intense protein synthesis in the Mn deficient chloroplasts. Hill activity of the –Mn leaves was about one-third of the analog control leaves. Manganese infiltration into detached but intact leaves restored the activity in the –Mn leaves up to 70% of the control. Lemon leaves affected by other macro- and micro- nutrient deficiencies did not respond to the manganese infiltration; therefore, the use of this infiltration method is suggested for the evaluation of the manganese nutrition status of citrus and probably other higher plants.     

Glumatic-Oxaloacetic Transaminase in Leaves of Phosphorus-Deficient Citrus Plants

Phosphorus deficiency in citrus leaves resulted in reduced glutamic-oxaloacetic transaminase (GOT) activity and low pyridoxal-phosphate (PLP) content. GOT activity was estimated in crude enzyme extracts by spectrophotometry PLP content was detected colorimetrically in water-alcoholic extracts. K and Cu deficiencies increased; -N, -S and -Zn decreased and -Mg, -Fe and -Mn did not affect GOT activity in citrus leaves. Experiments were conducted to restore enzyme activity either by direct addition of PLP to the reaction mixture or by infiltration of PLP or KH₂PO₄ to detached, intact leaves. The infiltration was carried out in vacuo and the leaves were incubated on wet paper for 26 h, after which enzyme activity was estimated. Transamination activity of -P leaves more restored by PLP than by KH₂PO₄ treatments. In zinc-deficient leaves the enzyme activity was not restored by infiltration of KH₂PO₄.     

Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization)

The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.     

A Class II fructose-1,6-bisphosphate aldolase from a halophilic archaebacterium Haloferax mediterranei

Fructose-1,6-bisphosphate (FBP) aldolase (EC 4.1.2.13) was purified 97-fold from a halophilic archaebacterium Haloferax mediterranei, with a specific activity of 2.8. The enzyme was characterized as a Class II aldolase on the basis of its inhibition by EDTA and other metal chelators. The enzyme had a specific requirement for divalent metal Fe(2+) for activity. Sulfhydryl compounds enhanced aldolase activity.     

Iron-shortage-induced increase in citric acid content and reduction of cytosolic aconitase activity in Citrus fruit vesicles and calli

Aconitase, which catalyses the conversion of citrate into isocitrate, requires Fe for its activity. The yeast and animal enzyme loses its enzymatic activity under Fe shortage and binds to RNA of genes involved in Fe homeostasis, altering their expression. Thus, the enzyme provides a regulatory link between organic acid metabolism and Fe cellular status. Roots and leaves of Fe-deficient plants show induction in organic acids, especially citrate. Although no RNA-binding activity has been so far demonstrated for the plant aconitase, whether alternations in enzyme activity by Fe could play a role in this induction remain unanswered. This question was investigated in lemon fruit [ Citrus limon (L.) Burm var Eureka ], characterized by the accumulation of citrate to about 0.3 M in the juice vesicles cells (pulp). Calli and isolated juice vesicles showed two- to three-fold induction in citrate level when subjected to Fe shortage. The mRNA level of aconitase exhibited no changes under reduced Fe concentrations. Analysis of aconitase isozymes demonstrated that out of two aconitase isozymes, typically detected in citrus fruit, only the cytosolic form displayed a reduced activity under low Fe concentrations. Our data support the notion of a limited Fe-availability-induced reduction in cytosolic aconitase, resulting in a slower rate of citrate breakdown and a concomitant increase in citrate levels.     

Development of cytosol and chloroplast aldolases during germination of spinach seeds

The total activity of aldolase (EC 4.1.2.13) and the activities of cytosol and chloroplast aldolase were determined in seeds, cotyledons, primary leaves and secondary leaves of spinach (Spinacia oleracea L., cv. Monopa) during germination. Total aldolase activity in cotyledons increased from low levels to a low maximum in the dark after one week and to a high maximum in white light after three to four weeks and declined thereafter. The activity in primary and secondary leaves started to rise strongly from the 18th and 26th days, respectively, up to the 42nd day of germination. The levels of aldolase activity paralleled the development of leaf area, chlorophyll content and protein content per leaf except that the leaf area of cotyledons continued to increase steadily up to the 42nd day after the maximum of aldolase activity was reached. Resolution of cytosol- and chloroplast-specific isoenzymes by chromatography on diethylaminoethylcellulose indicated that in the light the cytosol enzyme represented approx. 8% of the total activity in cotyledons, primary and secondary leaves throughout germination, and the chloroplast enzyme represented the remaining 92%. Only in cotyledons of dark-grown seedlings was the cytosol aldolase between 25 and 50% of the total activity. Seeds contained almost exclusively a cytosol aldolase. In cotyledons the increase of total activity in the light was specifically the consequence of an increase in chloroplast aldolase while the cytosol aldolase was little affected by light. The light effect was mediated by phytochrome as demonstrated by classical induction and reversion experiments with red and far-red light and by continuous far-red light treatment.Abbreviation DEAE-cellulose diethylaminoethylcellulose     

Modulation of the catalytic activity of pyridoxal kinase by metallothionein

Pyridoxal kinase displays high catalytic activity in the presence of metallothionein. The apoprotein of metallothionein as well as the peptide LYS-CYS-THR-CYS-CYS-ALA exert a strong inhibitory effect upon pyridoxal kinase by sequestering free Zn ions. Several steps intervene in the process of pyridoxal kinase activation, i.e. binding of Zn ions by ATP and interaction of Zn-ATP with the enzyme; but direct interaction between metallothionein and pyridoxal kinase (protein association) could not be detected by emission anisotropy measurements. Since the concentration of free Zn++ in mammalian tissues is lower than 10(-9)M, it is postulated that the concentration of metallothionein regulates the catalytic activity of pyridoxal kinase. The mechanism of reconstitution of the metalloenzyme yeast aldolase in the presence of metallothionein was also investigated.     

NOCTURNAL THERMAL EXCHANGE OF CITRUS LEAVES

Turrell , F. M., S. W. Austin , and R. L. Perry . (U. California, Riverside & Los Angeles.) Nocturnal thermal exchange of citrus leaves. Amer. Jour. Bot. 49(2) : 97–109. Illus. 1962.—Cooling rates of leaves were measured with fine thermocouples inserted within the leaf laminae. From these rates, total thermal conductances were calculated for leaves of intact greenhouse-grown lemon cuttings, in the dark, in still air and moving air, and in open laboratory rooms of warm to freezing temperatures. Thermal conductances were also calculated for leaves of 4 commercial varieties of citrus picked from mature trees in the grove and measured in low light, in still air, in a microcosm at warm, constant temperatures. The total conductances were fractionated, first, by determining transpiration rates of detached leaves from both sources, in darkness and in still or moving air, through similar temperature ranges and humidities. From transpiration rates, transpiration conductances were calculated. Second fractions (radiation conductances) were calculated for lemon leaves from far-infrared reflectances; and the third fractions (free-convection conductances) were calculated by subtraction of the sum of the radiation and transpiration conductances from the total. A free-convection-conductance coefficient was calculated for lemon, and then applied to 3 other varieties of citrus for which infrared reflectances were unavailable, to obtain their free-convection conductances. These together with experimentally determined transpiration and total conductances permitted calculation of their radiation fractions. The conductances have been tested for 6 different measured microclimates in which the calculated leaf temperatures averaged ± 0.6 C of the measured temperatures, an error compatible with the precision of field temperature measurements. Total thermal conductances of lemon leaves were higher in both warm, still and warm moving air than in cold, whereas the radiation and free-convection fractions were about equal in still air. The transpiration fractions were very small in warm, still or warm moving air but negligible in cold. In cold still and cold moving air, all the conductances were larger for orange fruit than for lemon leaves. Leaves of plants native to tropical rain forests were more efficient in heat transfer than were leaves from the temperate zone.     

Two Distinct Aldolases of Class II Type in the Cyanoplasts and in the Cytosol of the Alga Cyanophora paradoxa

Two aldolases from the alga Cyanophora paradoxa (Glaucocystophyta) can be separated by chromatography on diethylaminoethyl-Fractogel. The two aldolases are inhibited by 1 mM ethylene-diaminetetraacetate (EDTA) and, therefore, are class II aldolases. When cells of C. paradoxa were fractionated, one aldolase was associated with the cytosol fraction and the other was associated with the cyanoplast fraction. The Km(fructose-1,6-bisphosphate) was 600 [mu]M for the cytosolic aldolase and 340 [mu]M for the cyanoplast aldolase. The activity of the cytosolic aldolase was increased up to 4-fold by 100 mM K+ and slightly inhibited by Li+ and Cs+, whereas the cyanoplast aldolase was not affected by these ions. Inactivation by 1 mM EDTA could be partly restored by the addition of Co2+ or Mn2+ and to a lesser extent by Zn2+ or Mg2+. The molecular masses of the native cytosolic and cyanoplast aldolases are about 90 and 85 kD, respectively, as estimated by velocity centrifugation in sucrose gradients. Implications for the evolution of class I and II aldolases in chloroplasts of higher plants and algae will be discussed.     

Cloning and expression of cDNAs encoding plant V-ATPase subunits in the corresponding yeast null mutants

Complementation of yeast null mutants is widely used for cloning of homologous genes from heterologous sources. We have used this method to clone the relevant V-ATPase genes from lemon fruit and Arabidopsis thaliana cDNA libraries. The pH levels are very different in the vacuoles of the lemon fruit and the A. thaliana, yet both are the result of the activity of the same enzyme complex, namely the V-ATPase. In order to investigate the mechanism that enables the enzyme to maintain such differences in pH values, we have compared the subunit composition of the V-ATPase complex from both sources. Towards this end, we have constructed a cDNA library from lemon fruit and cloned it into a similar shuttle vector to the one of the A. thaliana cDNA library, which is commercially available. In this work, we report the cloning and expression of VMA10 from both sources, two isoforms of the lemon proteolipid (VMA3) and the lemon homologue of yeast VPH1/STV1 subunit, LEMAC.     

DNA-dependent RNA polymerase II from Candida species is a multiple zinc-containing metalloenzyme

We have purified DNA-dependent RNA polymerase II from Candida albicans, a human pathogenic yeast. The enzyme consists of 9 polypeptides that are unique to C. albicans, their mobility on SDS-PAGE being different from the mobility of the corresponding subunits of RNA polymerase II from Saccharomyces cerevisiae or C. utilis. In the present study we also demonstrate that RNA pol II from C. albican and C. utilis are metalloproteins containing approximately 5 mol of zinc per mole of enzyme. Although prolonged dialysis in 10 or 20 mM EDTA failed to remove Zn(II) from the C. albicans enzyme, in the C. utilis enzyme 3 Zn(II) ions could be removed and then reconstituted in the presence of excess Zn(II). o-Phenanthroline (5 mM) removed Zn(II) from C. albicans enzyme irreversibly in a time-dependent fashion with concomitant loss of enzyme activity. Circular dichroism studies revealed structural changes on removal of zinc, thus suggesting a role for Zn in maintenance of structural stability. Further, we demonstrate that the largest subunit of the C. utilis enzyme and the 3 large subunits of the C. albicans enzyme can bind radioactive zinc.     

Purification, subunit structure and immunological comparison of fructose-bisphosphate aldolases from spinach and corn leaves

The cytosol and chloroplast fructose-bisphosphate aldolases from spinach leaves were separated by ion-exchange chromatography on DEAE-cellulose, and were purified by subsequent affinity chromatography on phosphocellulose to apparent homogeneity as judged from polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The two aldolases had specific activities of 7.2 and 7.8 units mg protein-1. Molecular weight determinations by electrophoresis in sodium dodecyl sulfate gels and by sedimentation velocity centrifugation in sucrose gradients showed that the aldolases contained four subunits of Mr 38 000 and 35 000, respectively. Antibodies against the cytosol and chloroplast aldolase from spinach leaves were raised in a guinea pig and in a rabbit, respectively. In the Ouchterlony double-diffusion test, the two aldolases did not cross-react. A small degree of cross-reaction was observed by a test in which immune complexes were adsorbed to a solid-phase support (Staphylococcus aureus Cowan I cells) and nonbound enzyme activity was determined after centrifugation. These results imply major structural differences between the two spinach leaf aldolases. Only one major aldolase could be resolved on DEAE-cellulose from corn leaves. The aldolase was purified and had a specific activity of 6.4 units X mg protein-1. The corn leaf aldolase cross-reacted with the antiserum raised against the chloroplast enzyme from spinach leaves, but not with the other antiserum. Thus, the corn leaf aldolase could be identified as a chloroplast enzyme. Since aldolase activity is mostly restricted to the bundle sheath cells of corn leaf, it was concluded that it is compartmentalized in the chloroplasts of these cells but not in chloroplasts of the mesophyll cells.     

Protopectinase production in solid state culture ofAspergillus awamori

Summary Protopectinases (PPases) are a heterogeneous group of enzymes that release water soluble pectin from insoluble protopectin in plant tissues by restricted degradation of the substrate. In all cases reported to date, PPases of bacterial or yeast origin were produced in liquid culture. Here, we describe the growth and PPase production ofAspergillus awamori IFO 4033 in solid state culture. Petri dishes containing 10 g of wheat bran and 15 ml of 0.2 M HCl were inoculated with 2 ml of a suspension with 1 × 10⁵ spores.ml⁻¹ and incubated for 48 h at 30°C. PPase activity on lemon (PPase-l) and apple (PPase-a) protopectins was maximum at 24 h of culture (1490 and 610 U.g⁻¹, respectively) and then decreased. Pectinase activity on lemon and apple pectin and polygalacturonase activity were maximum at 48 h. Hence, the crude enzyme pool obtained at 24 h of process was appropriate for extraction of citrus and apple pectin with a minor subsequent degradation of the solubilized pectin. The ratio of PPase-l to PPase-a changed during culture, so there seemed to be at least two PPases with different substrate specificity.     

Purification and characterization of the class-II D-fructose 1,6-bisphosphate aldolase from Escherichia coli (Crookes'' strain).

A new form of the class-II D-fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) of Escherichia coli (Crookes' strain) was isolated from an extract of glycerol-grown bacteria. It has a higher molecular weight (approx. 80000)than previous preparations of the enzyme and closely resembles the typical class-II aldolase from yeast in size and amino acid composition. On the other hand, its kinetic behaviour is not typical of a class-II aldolase. The enzyme has no requirement for thiol compounds either for stability or activity, added K+ ions have no effect, and the optimum pH for the cleavage activity is unusually high. The class-II enzymes from the prokaryote E. coli and the eukaryote yeast show no immunological identity. However, the similarity of their structures suggests that they have evolved from a common ancestor.     

A zinc-binding citrus protein metallothionein can act as a plant defense factor by controlling host-selective ACR-toxin production

Metallothionein is a small cysteine-rich protein known to have a metal-binding function. We isolated three different lengths of rough lemon cDNAs encoding a metallothionein (RlemMT1, RlemMT2 and RlemMT3), and only RlemMT1-recombinant protein had zinc-binding activity. Appropriate concentration of zinc is an essential micronutrient for living organisms, while excess zinc is toxic. Zinc also stimulates the production of host-selective ACR-toxin for citrus leaf spot pathogen of Alternaria alternata rough lemon pathotype. Trapping of zinc by RlemMT1-recombinant protein or by a zinc-scavenging agent in the culture medium caused suppression of ACR-toxin production by the fungus. Since ACR-toxin is the disease determinant for A. alternata rough lemon pathotype, addition of RlemMT1 to the inoculum suspension led to a significant decrease in symptoms on rough lemon leaves as a result of reduced ACR-toxin production from the zinc trap around infection sites. RlemMT1-overexpression mutant of A. alternata rough lemon pathotype also produced less ACR-toxin and reduced virulence on rough lemon. This suppression was caused by an interruption of zinc absorption by cells from the trapping of the mineral by RlemMT1 and an excess supplement of ZnSO₄ restored toxin production and pathogenicity. Based on these results, we propose that zinc adsorbents including metallothionein likely can act as a plant defense factor by controlling toxin biosynthesis via inhibition of zinc absorption by the pathogen.     

Overexpression of citrus polygalacturonase-inhibiting protein in citrus black rot pathogen Alternaria citri

The rough lemon (Citrus jambhiri) gene encoding polygalacturonase-inhibiting protein (RlemPGIPA) was overexpressed in the pathogenic fungus Alternaria citri. The overexpression mutant AcOPI6 retained the ability to utilize pectin as a sole carbon source, and the overexpression of polygalacturonase-inhibiting protein did not have any effect on the growth of AcOPI6 in potato dextrose and pectin medium. The pathogenicity of AcOPI6 to cause a black rot symptom in citrus fruits was also unchanged. Polygalacturonase-inhibiting protein was secreted together with endopolygalacturonase into culture filtrates of AcOPI6, and oligogalacturonides were digested from polygalacturonic acid by both proteins in the culture filtrates. The reaction mixture containing oligogalacturonides possessed activity for induction of defense-related gene, RlemLOX, in rough lemon leaves.     

Identification and characterization of a heat-induced isoform of aldolase in oat chloroplast

An analysis of protein synthesis at elevated temperatures in oat (Avena sativa) leaves revealed a heat-induced 44 kDa polypeptide. A cDNA library of heat-treated leaves was constructed and screened with specific antibodies raised against this 44 kDa polypeptide. A clone encoding the 44 kDa protein was identified as a form of the chloroplast-localized fructose-bisphosphate aldolase (EC 4.1.2.13). Northern and western blot analyses indicated heat-induced accumulation of the chloroplast aldolase isoform at both the RNA and protein level. Heat inducibility was restricted to the chloroplastic form of the enzyme, and was not observed for the cytoplasmic aldolase. The heat-induced isoform co-purified with thykaloid fractions, as confirmed by immunoassay and activity analyses. However, when thylakoid membranes were treated with proteinase K, the aldolase isoform completely disappeared, suggesting that this enzyme is not embedded but rather tends to adhere to the chloroplast membranes. Immunoblot analysis of other plant species revealed similar heat induction of thykaloid-associated aldolase homologues, suggesting the possible existence of a universal control mechanism for this enzyme's heat tolerance