Chlorotic Plants Research Articles

Organogenesis of Plant Tissues in Colchicine Allows Selecting in Field Trial Blueberry (Vaccinium spp. cv Duke) Clones with Commercial Potential

Plants’ genetic improvement continues to be crucial for modern agriculture, while biotechnology can offer efficient tools that enhance the selection and recommendation processes of elite clones. This work established a suitable methodology for the regeneration of blueberry (Vaccinium corymbsum) plants in cultures with colchicine. This could be considered a basis for producing populations for the selection of clones following a genetic improvement program assisted by biotechnology. The factors studied were: (a) explant type (leaf discs; nodal segments); (b) colchicine concentration (0, 0.5, 1, and 2 mg/L); and (c) time of exposure to colchicine (1, 2, 3, 5, and 30 days). The basal medium McCown’s Woody Plant (WP) supplemented with 2 mg/L 2iP and 1 mg/L BAP was used with the commercial genotype Duke as a model. A total of 1957 blueberry clones were produced in a medium with 1 mg/L colchicine, distributed at different exposure times. Flow cytometry analyses revealed the following patterns: single patterns for random samples of control plants (Duke donor) and some clones regenerated on colchicine; double patterns for chlorotic plants regenerated on colchicine. Triple and quadruple patterns were observed in callus tissues that did not regenerate plants on colchicine. Populations of plants regenerated in colchicine (6787) and control plants regenerated in in vitro culture without colchicine were adapted under greenhouse conditions. The variables evaluated at this stage were adaptability, height, diameter, number of leaves, incidence of diseases, flowering capacity, and agrobotanical traits. Selected clones demonstrating phenotypic variability (157 clones) were transplanted to field conditions. From the clonal field trial conducted under minimum tillage conditions, 38 clones were selected for improved traits related to the agricultural yield and nutritional quality of the fruits. Of these, six clones showed the highest agronomic performance and adaptability to adverse environmental conditions compared to the Duke donor genotype. It is recommended that these clones continue genotype × environment interaction trials at different locations.

First report of Fusarium verticillioides causing safflower root rot in Sinaloa, Mexico.

Safflower cultivation is of great socioeconomic importance worldwide. Production is intended for the extraction of oil from the seeds. In 2021 Mexico ranked fifth in world production with approximately 52,553.28 tons (SIAP, 2021). In April 2022, in the north-central zone of Sinaloa, Mexico, diseased plants were reported in fields planted with safflower. Symptoms included chlorotic plants, necrosis and rot in vascular bundles, dwarfed plants and reflexed plants bent towards the ground. The disease caused estimated losses of 15% of seed production, with respect to the production obtained from the previous year in the safflower fields surveyed. Twenty-five plants with symptoms were sampled to isolate the pathogen. Plants were cut at the base of the stem near the roots and roots cut into 5 mm2 pieces. Tissue samples were superficially disinfected by immersing in 70% alcohol for 10 sec, 2% sodium hypochlorite for 1 min, washed in sterile water, and placed on potato dextrose agar (PDA) at 28 ºC for 7 days in the dark. Twelve monosporic isolates derived from the PDA culture were morphologically characterized. Abundant white aerial mycelium and small pink to dark violet pigments in the center of the culture were observed. From 10-day-old cultures grown on carnation leaf agar medium microconidia and macroconidia were produced. Microconidia were hyaline, had zero to two septa, and were oval or ellipsoidal, 4.6 to 14 x 1.8 to 4.2 μm (n = 40). The macroconidia were hyaline, were slightly curved with three to five septa, and measured from 26 to 69 x 3 to 6.1 μm (n = 40). No chlamydospores were observed. According to the morphological characteristics, the isolates were identified as Fusarium verticillioides (Leslie and Summerell, 2006). DNA was extracted from one isolate and the Translation Elongation Factor 1-α (EF1) gene was amplified and sequenced (O'Donnell et al. 2010). The sequence obtained from isolate FV3CARCULSIN with 645 base pairs was submitted to NCBI GenBank with accession number OQ262963. The BLAST search revealed 100% similarity with F. verticillioides isolate 13 (KM598773) (Lizárraga et al. 2015). Identification in FUSARIUM ID resulted in a 99.85% similarity with isolate F. verticillioides CBS 131389 (MN534047) (Yilmaz et al. 2021). A phylogenetic tree, made with sequences of the EF1 gene, revealed that FV3CARCULSIN was most closely related to F. verticillioides (100% bootstrap). Pathogenicity tests were carried out on safflower plants (cv. Oleico) grown in sterile vermiculite. Plants were inoculated with a conidial suspension (1 × 105 conidia/ml) obtained from FV3CARCULSIN grown on PDA for 7 days. A total of 45 plants were inoculated by drenching the roots with 20 ml of inoculum when the plants were 20 days old. Fifteen plants served as negative controls without inoculation. Plants were kept for 60 days in greenhouse conditions; however, after 45 days the plants began to die. The assay was conducted twice. Rotting and necrosis was observed in the roots of the plants. The pathogen was reisolated from the tissue of all the plants with symptoms and identified as F. verticillioides using morphological characteristics and EF1 sequences, completing Koch's postulates. No symptoms were observed in control plants after 60 days. This is the first report of root rot in safflower caused by F. verticillioides in Mexico. The fungus has been reported in maize (Figueroa et al. 2010), but it is unknown if it could be the same pathogen of safflower. Identification of the pathogen is important for implementing management methods to reduce yield losses and for additional studies on the impact of the disease on oil quality extracted from safflower seeds.

Metagenomic analysis of rhizosphere microbiome provides insights into occurrence of iron deficiency chlorosis in field of Asian pears

BackgroundFe-deficiency chlorosis (FDC) of Asian pear plants is widespread, but little is known about the association between the microbial communities in the rhizosphere soil and leaf chlorosis. The leaf mineral concentration, leaf subcellular structure, soil physiochemical properties, and bacterial species community and distribution had been analysed to gain insights into the FDC in Asian pear plant.ResultsThe total Fe in leaves with Fe-deficiency was positively correlated with total K, Mg, S, Cu, Zn, Mo and Cl contents, but no differences of available Fe (AFe) were detected between the rhizosphere soil of chlorotic and normal plants. Degraded ribosomes and degraded thylakloid stacks in chloroplast were observed in chlorotic leaves. The annotated microbiome indicated that there were 5 kingdoms, 52 phyla, 94 classes, 206 orders, 404 families, 1,161 genera, and 3,043 species in the rhizosphere soil of chlorotic plants; it was one phylum less and one order, 11 families, 59 genera, and 313 species more than in that of normal plant. Bacterial community and distribution patterns in the rhizosphere soil of chlorotic plants were distinct from those of normal plants and the relative abundance and microbiome diversity were more stable in the rhizosphere soils of normal than in chlorotic plants. Three (Nitrospira defluvii, Gemmatirosa kalamazoonesis, and Sulfuricella denitrificans) of the top five species (N. defluvii, G. kalamazoonesis, S. denitrificans, Candidatus Nitrosoarchaeum koreensis, and Candidatus Koribacter versatilis). were the identical and aerobic in both rhizosphere soils, but their relative abundance decreased by 48, 37, and 22%, respectively, and two of them (G. aurantiaca and Ca. S. usitatus) were substituted by an ammonia-oxidizing soil archaeon, Ca. N. koreensis and a nitrite and nitrate reduction related species, Ca. K. versatilis in that of chlorotic plants, which indicated the adverse soil aeration in the rhizosphere soil of chlorotic plants. A water-impermeable tables was found to reduce the soil aeration, inhibit root growth, and cause some absorption root death from infection by Fusarium solani.ConclusionsIt was waterlogging or/and poor drainage of the soil may inhibit Fe uptake not the amounts of AFe in the rhizosphere soil of chlorotic plants that caused FDC in this study.

A novel plant extract as a biostimulant to recover strawberry plants from iron chlorosis

The traditional agricultural production systems are evolving more toward organic, sustainable or environmental friendly systems and products; however, it is important to keep the yield and the quality of crops. The aim of this work was to evaluate the recovery of iron deficient strawberry plants by foliar spray, using a gramineous plants extract (GE). Bare-root transplants of strawberry (Fragaria × ananassa Duch. cv “Diamond”) were grown in a Hoagland’s nutrient solution with iron (Fe10) or without (Fe0). Forty-two days after Fe deprivation, plants grown without iron became chlorotic and GE was applied. Three foliar applications were done, once per week and the regreening was monitored using a SPAD apparatus. After two applications, a rapid but transient regreening was detected in young leaves. Nevertheless, biomass and root/shoot ratio of recovered plants (GE treatment) were similar to chlorotic plants (Fe0 treatment). Phosphorous, Mn and Fe contents were higher in crowns of GE plants at the end of the experiment (67 days). The concentration of organic acids and ferric chelate–reductase (FC-R) activity was also significantly higher in GE treatment. It is possible that the high chelating capacity of the extract was responsible for the Fe internal transport and use.

Physio-morphological and biochemical responses of pot marigold (Calendula officinalis L.) to split iron nutrition

The study was aimed to determine the iron (Fe) requirements at different growing stages of pot marigold (Calendula officinalis L.). Plants were grown in pots containing equal proportions of sand and perlite, and fertigated with nutrient solution including 4 or 35 µM of the Fe-chelate Fe(III)-EDDHA in each of three different growth stages. Stages were labeled I (from seed germination until 4th to 6th true leaf stage), II (from 4th to 6th true leaf stage until start of flowering) and III (from the start of flowering to flower fading time). Using consistently 4 µM Fe led to chlorotic plants and small and short-lived flowers, whereas using consistently 35 µM Fe led to good quality flowers, considering size, number and lifetime. Results suggest that using 4 µM Fe at growth stages I or III did not result in significant reductions in flower weight and number. Conversely, using 4 µM Fe at stage II led to decreases in flower weight and number. Using 4 µM Fe in two stages could be acceptable only when stages were I and III, although some decreases in number of flowers and number longevity occurred. Results show that it is feasible to reduce the concentration of Fe in the nutrient solution during a considerable part of the plant growth period. Using low Fe in only one growth stage would reduce total Fe inputs by 20–29%, whereas using low Fe in stages I and III would reduce total Fe inputs by 48%. The reduction proposed in the Fe-chelate concentrations would result in diminishing costs for growers and decreasing the environmental impact of Fe fertilization.

First Report of Ceratobasidium sp. Causing Root Rot of Garlic in China

Garlic (Allium sativum L.) is an important cash crop in China. In the spring of 2018, stunted and chlorotic plants in some garlic fields were observed in Jinxiang, Shandong, and Feng Counties, Jiangsu Province, in China. The growth of symptomatic plants was weaker, with affected plants showing signs of apical chlorosis, wilting, and dieback. In the aerial part of the plant, the symptoms of root rot disease resembled nutrient deficiency or drought stress. The roots were shriveled and rotted, with reduced root density. According to survey, the incidence of root rot in each garlic planting area is as high as 35 to 40%, and once the disease occurs, the commodity value of garlic will be reduced. Generally speaking, fungal diseases are among the main biotic factors affecting garlic yield, particularly those caused by species of the genera Fusarium, Phytophthora, and Pythium. In order to identify the causal agent of this disease, symptomatic plants were collected from several regions of the main garlic producing areas in China, including Jinxiang County, Jiaxiang County in Shandong Province, and Fengxian County in Jiangsu Province. The garlic variety planted is Jinxiang white garlic, which is a local garlic variety. Samples of rotten roots were washed in tap water, surface sterilized in 1.5% sodium hypochlorite for 1 min followed by 75% ethanol for 30 s, rinsed with sterilized distilled water three times, and blotted dry on sterile filter paper. Pieces of infected root tissues (2 to 3 mm) were placed on potato dextrose agar (PDA) and kept at 28°C for 7 days. After the colonies were established on PDA, the fungal strains were purified by the hyphal-tip method. Five fungal isolates with similar morphology to each other were obtained, and isolate DS54-3 was chosen for the following experiment. The fungal colony grew within 5 days on PDA containing a full culture medium. Fungal colonies were white initially and then turned brown, and septate hyphae were 3.7 to 4.3 μm in diameter and branched at right angles with a constriction at the origin of the branch point. These characteristics are typical of the genus Rhizoctonia. Binucleate cells from five isolates were observed using a lactophenol aniline blue solution stain and matched Ceratobasidium morphological descriptions (Chen et al. 1985). The morphological characteristics of isolate DS54-3 were consistent with Ceratobasidium sp. (Sneh et al. 1991). Mycelia from five isolates grown on PDA were used for DNA extraction. The rDNA-ITS region was amplified using PCR with the universal fungal primers ITS1 and ITS4 (White et al. 1990). The purified products were separately sequenced in both directions using the same primer pair (Meza-Moller et al. 2014). ITS sequence analysis (GenBank accession no. MK733974) resulted in a 100% match for one accession of Ceratobasidium sp. AG-A (MF070683.1) by BLAST in the NCBI nucleotide database. The pathogenicity of isolate DS54-3 was then tested. For each conidial suspension,12 healthy 10-day-old garlic plants (about 5 cm) were used to conduct pathogenicity tests by wound inoculation, and the garlic varieties were the same as those collected in the field. The healthy garlic plants were inoculated with 5, 10, 20, 50, and 100 ml of conidial suspension (2.95 × 10⁷ conidia/ml) by the root irrigation method. Twenty plants were inoculated with the same volume of sterile water as a control for each conidial suspension. After 20 days, the leaves of all garlic seedlings inoculated with conidial suspension gradually turned yellow, similar to the symptoms observed during natural infection. After 40 days, the root system showed typical hollow and rotten symptoms, and eventually the plants withered and died, whereas the control plants remained healthy. The same colonial fungus was reisolated, thus fulfilling Koch’s postulates. This is the first report of Ceratobasidium sp. causing root rot disease of garlic in China.

Differences in Aceria tosichella population responses to wheat resistance genes and wheat virus transmission

Severe winter wheat yield losses due to infestations of wheat curl mite, Aceria tosichella Keifer, and mite-transmitted viruses occur in wheat production areas of the United States and Canada. Mite infestation alone causes stunted, chlorotic plants in susceptible wheat varieties, and mites transmit Wheat Streak Mosaic (WSMV), High Plains Wheat Mosaic (HPWMoV), and Triticum Mosaic Virus (TriMV). Wheat curl mites were collected from 25 sites in Kansas, Missouri, Nebraska, Texas, North Dakota, and South Dakota in 2014 and 2015. At each site, mite virulence was determined to wheat plants harboring the Cmc2-, Cmc3-, or Cmc4 mite resistance gene; or Cmc4 plus the Wsm2 WSMV resistance gene. Mites collected from 92%, 36%, and 24% of sites were virulent to susceptible Jagger wheat plants (no Cmc), Cmc2, and Cmc3, respectively. The mega-population consisting of all 25 mite sub-populations was avirulent to 80% of plants containing Cmc4 + Wsm2 or Cmc4. WSMV, HPWMoV, or TriMV was present in mites at 76%, 16%, and 8% of the 25 sites, respectively. Our results will enable breeders to increase the efficiency of wheat production by releasing wheat varieties containing wheat curl mite resistance genes that reduce wheat yield losses.

First Report of Setophoma terrestris Causing Pink Root of Garlic in China

Garlic (Allium sativum L.) is an important crop in China. In the spring of 2015, stunted and chlorotic plants in some garlic crops were observed in Jinxiang, Shandong Province, and Feng County, Jiangsu Province, in China. The growth of field symptomatic plants was weaker, with affected plants showing signs of apical chlorosis, wilting, and dieback. In the aerial part of the plant, the symptoms of pink root disease resembled nutrient deficiency or drought. The roots were pink and rotted, with reduced root density. To identify the causal agent of this disease, symptomatic plants were sampled. Samples of pink roots were washed in tap water, surface sterilized in 1.5% sodium hypochlorite for 1 min and then 75% ethanol for 30 s, rinsed with sterilized distilled water three times, and then blotted dry on sterile filter paper. Pieces of infected root tissues (2 to 3 mm) were cut and placed on potato dextrose agar (PDA) at 28°C for 7 days. After the colonies were established on PDA, the fungal strains were purified by the hyphal-tip method. Eight fungal isolates were obtained, and they were morphologically similar to each other, so H5 was chosen for the following experiment. Its colony grew slowly (5 cm in 10 days) on PDA plates, with abundant aerial mycelium, which were initially pale pink but changed to dark red with growth. Microconidia were not observed on PDA plates. For sporulation and pycnidia generation, isolate H5 was cultured on corn meal agar at 28°C under darkness for 7 days and then removed to combined 13-h photoperiod and 11-h darkness conditions for 7 days. It produced dark brown pycnidia (globose to subglobose, 160 to 230 µm) with brown setae 55 to 150 µm long. Pycnidiospores (4.0 to 5.0 × 1.5 to 2.0 µm) were oblong ovoid and oozed from rupture or through the ostiole. The morphological characteristics of H5 were consistent with S. terrestris (Boerema et al. 2004). Total genomic DNA was extracted from mycelium of isolate H5, and the large subunit region was amplified by polymerase chain reaction with the primer pair LROR and LR5 (de Gruyter et al. 2010). The sequence of isolate H5 was deposited in GenBank (MH046778), and a BLASTn search showed 100% similarity with several Setophoma terrestris sequences (GQ387587). The pathogenicity of isolate H5 was tested. Twelve healthy 10-day-old garlic sprouts (about 5 cm) were used to conduct pathogenicity tests by wound inoculation. Nine plants were inoculated with 10 ml of conidial suspension (1.2 × 10⁵ conidia/ml), respectively, by the root irrigation method, and another three plants were inoculated with the same volume of sterile water as a control. After 40 days, the leaves gradually yellowed, similar to leaf symptoms observed during natural infection. The roots showed typical symptoms of pink root, and finally the whole plant wilted and died, whereas the control plants remained healthy. The same colonial fungus was reisolated, thus fulfilling Koch’s postulates. The pathogen associated with pink root of various vegetables was hitherto considered to be Pyrenochaeta terrestris (H.N. Hansen, Gorenz, J.C. Walker & Larson or Phoma terrestris H.N. Hansen). To our knowledge, this is the first report of S. terrestris causing pink root of garlic in China.

Phytotoxic effects of Morus nigra aqueous extract on germination and seedling growth of Lactuca sativa

Abstract Some exotic species threat the integrity of natural environments due to their invasive potential. They can affect other species by releasing secondary compounds in the soil. Morus nigra (Moraceae) is an invasive species of riparian forests in southern Brazil. The objective of this study was to verify if the aqueous extracts of fruit, fresh and dry leaves of M. nigra show phytotoxic effects on germination, seedling growth and membrane integrity of seedlings of Lactuca sativa. Extract concentrations of 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5 and 10% were tested. Germination rate and speed of germination were determined. Effects on initial growth were evaluated by measuring seedling root and shoot length, and membrane integrity was assessed by conductivity tests. Results showed the phytotoxicity of M. nigra aqueous extracts, causing significant inhibition on germination and seedling growth. The fruit extract was generally less phytotoxic than extracts from fresh and dry leaves. Moreover, the extracts induced changes in membrane integrity and caused morphological deformities in seedlings, such as necrotic roots and chlorotic plants. The high phytotoxicity of fruit, dry and fresh leaf extracts of M. nigra was evidenced, indicating the allelopathic potential of the species.

AtRsgA from Arabidopsis thaliana is important for maturation of the small subunit of the chloroplast ribosome.

Plastid ribosomes are very similar in structure and function to the ribosomes of their bacterial ancestors. Since ribosome biogenesis is not thermodynamically favorable under biological conditions it requires the activity of many assembly factors. Here we have characterized a homolog of bacterial RsgA in Arabidopsis thaliana and show that it can complement the bacterial homolog. Functional characterization of a strong mutant in Arabidopsis revealed that the protein is essential for plant viability, while a weak mutant produced dwarf, chlorotic plants that incorporated immature pre-16S ribosomal RNA into translating ribosomes. Physiological analysis of the mutant plants revealed smaller, but more numerous, chloroplasts in the mesophyll cells, reduction of chlorophyll a and b, depletion of proplastids from the rib meristem and decreased photosynthetic electron transport rate and efficiency. Comparative RNA sequencing and proteomic analysis of the weak mutant and wild-type plants revealed that various biotic stress-related, transcriptional regulation and post-transcriptional modification pathways were repressed in the mutant. Intriguingly, while nuclear- and chloroplast-encoded photosynthesis-related proteins were less abundant in the mutant, the corresponding transcripts were increased, suggesting an elaborate compensatory mechanism, potentially via differentially active retrograde signaling pathways. To conclude, this study reveals a chloroplast ribosome assembly factor and outlines the transcriptomic and proteomic responses of the compensatory mechanism activated during decreased chloroplast function.

Azotochelin and N-dihydroxy-N,N’-diisopropylhexanediamide as Fe sources to cucumber plants in hydroponic cultures

Environmental concerns related to the use of synthetic iron chelates, usually non-biodegradable, for overcoming iron chlorosis motivates the search for alternative compounds. Thus, the main aim of this work was to evaluate siderophore, azotochelin, and a siderophore mimic, N-dihydroxy-N,N’-diisopropylhexanediamide (DPH) as potential sources of iron to cucumber plants grown in hydroponic cultures. The behavior of the iron chelates of azotochelin and DPH, as a substrate of ferric chelate reductase (FCR) and the ability as iron suppliers for chlorotic plants was studied and compared with o,o-EDDHA/Fe3+ and EDTA/Fe3+ chelates, traditionally used for this purpose. The rate of reduction of DPH/Fe3+, by FCR, was comparable to o,o-EDDHA/Fe3+ but lesser than the obtained for EDTA/Fe+3. The rate of reduction for azotochelin/Fe3+ was not possible to determine. Both azotochelin/Fe3+ and DPH/Fe3+ chelates were effective in supplying iron to cucumber plants. After 7 and 21 days, all the plants treated with the iron chelates (10 µM, Fe) of DPH and azotochelin showed significantly higher SPAD index, leaf dry weight and leaf Fe concentration than the control plants (2µM, Fe). In conclusion, azotochelin/Fe3+ and DPH/Fe3+ can be considered as iron sources for cucumber plants when growing in hydroponic culture.

Silencing of the FRO1 gene and its effects on iron partition in Nicotiana benthamiana

To evaluate the dynamic role of the ferric-chelate reductase enzyme (FCR) and to identify possible pathways of regulation of its activity in different plant organs an investigation was conducted by virus-induced gene silencing (VIGS) using tobacco rattle virus (TRV) to silence the ferric reductase oxidase gene (FRO1) that encodes the FCR enzyme. Half of Nicotiana benthamiana plants received the VIGS vector and the rest remained as control. Four treatments were imposed: two levels of Fe in the nutrient solution (0 or 2.5 μM of Fe), each one with silenced or non-silenced (VIGS-0; VIGS-2.5) plants. Plants grown without iron (0; VIGS-0) developed typical symptoms of iron deficiency in the youngest leaves. To prove that FRO1 silencing had occurred, resupply of Fe (R) was done by adding 2.5 μM of Fe to the nutrient solution in a subset of chlorotic plants (0-R; VIGS-R). Twelve days after resupply, 0-R plants had recovered from Fe deficiency while plants containing the VIGS vector (VIGS-R) remained chlorotic and both FRO1 gene expression and FCR activity were considerably reduced, consequently preventing Fe uptake. With the VIGS technique we were able to silence the FRO1 gene in N. benthamiana and point out its importance in chlorophyll synthesis and Fe partition.

Transcriptional Characterization of a Widely-Used Grapevine Rootstock Genotype under Different Iron-Limited Conditions.

Iron chlorosis is a serious deficiency that affects orchards and vineyards reducing quality and yield production. Chlorotic plants show abnormal photosynthesis and yellowing shoots. In grapevine iron uptake and homeostasis are most likely controlled by a mechanism known as “Strategy I,” characteristic of non-graminaceous plants and based on a system of soil acidification, iron reduction and transporter-mediated uptake. Nowadays, grafting of varieties of economic interest on tolerant rootstocks is widely used practice against many biotic and abiotic stresses. Nevertheless, many interspecific rootstocks, and in particular those obtained by crossing exclusively non-vinifera genotypes, can show limited nutrient uptake and transport, in particular for what concerns iron. In the present study, 101.14, a commonly used rootstock characterized by susceptibility to iron chlorosis was subjected to both Fe-absence and Fe-limiting conditions. Grapevine plantlets were grown in control, Fe-deprived, and bicarbonate-supplemented hydroponic solutions. Whole transcriptome analyses, via mRNA-Seq, were performed on root apices of stressed and unstressed plants. Analysis of differentially expressed genes (DEGs) confirmed that Strategy I is the mechanism responsible for iron uptake in grapevine, since many orthologs genes to the Arabidopsis “ferrome” were differentially regulated in stressed plant. Molecular differences in the plant responses to Fe absence and presence of bicarbonate were also identified indicating the two treatments are able to induce response-mechanisms only partially overlapping. Finally, we measured the expression of a subset of genes differentially expressed in 101.14 (such as IRT1, FERRITIN1, bHLH38/39) or known to be fundamental in the “strategy I” mechanism (AHA2 and FRO2) also in a tolerant rootstock (M1) finding important differences which could be responsible for the different degrees of tolerance observed.

Effectiveness of Iron Ethylenediamine-N,N'-bis(hydroxyphenylacetic) Acid (o,o-EDDHA/Fe3+) Formulations with Different Ratios of Meso and d,l-Racemic Isomers as Iron Fertilizers.

Two o,o-EDDHA/Fe3+ formulations (meso, 93.5% w/w of meso isomer; and d,l-racemic, 91.3% w/w of d,l-racemic mixture) were prepared, and their efficacy to avoid or to relieve iron deficiency in Fe-sufficient and Fe-deficient tomato plants grown on hydroponic solution was compared with that of the current o,o-EDDHA/Fe3+ formulations (50% of meso and d,l-racemic isomers). The effectiveness of the three o,o-EDDHA/Fe3+ formulations was different depending on the iron nutritional status of plants. The three o,o-EDDHA/Fe3+ formulations tested were effective in preventing iron chlorosis in healthy plants. However, the higher the meso concentration in the formulations, the higher the effectiveness in the recovery of iron chlorotic plants from iron deficiency. Accordingly, o,o-EDDHA/Fe3+ formulations rich in meso isomer are recommended in hydroponic systems.

The catalytic subunit of magnesium-protoporphyrin IX monomethyl ester cyclase forms a chloroplast complex to regulate chlorophyll biosynthesis in rice.

YGL8 has the dual functions in Chl biosynthesis: one as a catalytic subunit of MgPME cyclase, the other as a core component of FLU-YGL8-LCAA-POR complex in Chl biosynthesis. Magnesium-protoporphyrin IX monomethyl ester (MgPME) cyclase is an essential enzyme involved in chlorophyll (Chl) biosynthesis. However, its roles in regulating Chl biosynthesis are not fully explored. In this study, we isolated a rice mutant yellow-green leaf 8 (ygl8) that exhibited chlorosis phenotype with abnormal chloroplast development in young leaves. As the development of leaves, the chlorotic plants turned green accompanied by restorations in Chl content and chloroplast ultrastructure. Map-based cloning revealed that the ygl8 gene encodes a catalytic subunit of MgPME cyclase. The ygl8 mutation caused a conserved amino acid substitution (Asn182Ser), which was related to the alterations of Chl precursor content. YGL8 was constitutively expressed in various tissues, with more abundance in young leaves and panicles. Furthermore, we showed that expression levels of some nuclear genes associated with Chl biosynthesis were affected in both the ygl8 mutant and YGL8 RNA interference lines. By transient expression in rice protoplasts, we found that N-terminal 40 amino acid residues were enough to localize the YGL8 protein to chloroplast. In vivo experiments demonstrated a physical interaction between YGL8 and a rice chloroplast protein, low chlorophyll accumulation A (OsLCAA). Moreover, bimolecular fluorescence complementation assays revealed that YGL8 also interacted with the other two rice chloroplast proteins, viz. fluorescent (OsFLU1) and NADPH:protochlorophyllide oxidoreductase (OsPORB). These results provide new insights into the roles of YGL8, not only as a subunit with catalytic activity, but as a core component of FLU-YGL8-LCAA-POR complex required for Chl biosynthesis.

Fe deficiency induction in Poncirus trifoliata rootstock growing in nutrient solution changes its performance after transplant to soil

The absence of iron (Fe) in the nutrient solution induces several physiological and morphological adaptations in the roots of Poncirus trifoliata, a citrus rootstock, thereby modifying its overall nutritional status. Whether these changes are advantageous when plants are transplanted to calcareous soils needs to be assessed. To achieve this objective a two-phase experiment was established, first in nutrient solution (phase I) then in pots containing different soils (phase II). In phase I, P. trifoliata L. Raf. plants were grown in Hoagland's solution with 120μM of Fe (Fe120 treatment) or without (Fe0 treatment). At the end of phase I (87 days), Fe-chlorotic plants had less chlorophyll in apical younger leaves, root tips were swollen and their FC-R activity was enhanced, typical responses to Fe-stress. Chlorotic plants had less Fe compared to control plants, but accumulated more Cu and Zn. In contrast the root to shoot ratio (dry weight) and the amounts of macronutrients were not affected by Fe chlorosis. In phase II, plants of both treatments were transplanted to pots containing a calcareous (C) or a non-calcareous (nC) soil resulting in four treatments: Fe0nC, Fe120nC, Fe0C and Fe120C. From the end of phase I until the end of the experiment (353 days), the calcareous soil negatively affected the overall nutritional balance in both Fe0 and Fe120 treatments. Apparently, the ability to change metal homeostasis in particular Cu, as a Fe-stress response was maintained in plants grown in non-calcareous soil. Moreover, the previous induction of physiological and morphological adaptations to Fe depletion alleviated the iron chlorosis symptoms caused by soil carbonates. These results may point to the utilization of internal stress signalling as a tool to cope with different soil conditions.

Effect of iron deficiency stress on leaves movements and electrical potentials in mimosa (Mimosa pudica L.)

The aim of the studies was to trace the motorical and electrical activity of the mimosa (<em>Mimosa pudica</em> L.) grown under conditions of iron deficiency. The speed of leaf folding was measured (motorical activity) and the action potential induced with thermic and light stimuli and turgorin (electrical activity was recorded). It was found that the iron deficiency caused acidification of medium and the maximum of the process coincided with the period when the young leaves were turning green. Chlorotic mimosa leaves, not detached from the plant, showed an increased motorical activity. Motorical and electrical activity of the leaves were inhibited by an inhibitor of the plasmalemma redox systems - quinacrine, and stimulated by blue light. Leaf movement factor - turgorin - caused a hypersensitivity of chlorotic plants. It follows from the studies that the observed effects resulted from the adaptation of mimosa to the iron stress. The adaptation was a result of formation of new plasmalemma redox systems (turbo-reductase), responsible for maintaining high energy levels in the cells.

Quantification of ethylenediamine-N,N′-bis(hydroxysulfophenylacetic) acid regioisomers and structural characterisation of its related polycondensation products by porous graphitic carbon high-performance liquid chromatography coupled to electrospray tandem mass spectrometry

Among the commercial ethylenediamine-N,N′-bis(2-hydroxy)phenylacetic acid/iron(III) derivatives, ethylenediamine-N,N′-bis(2-hydroxy-5-sulphophenylacetic) acid/iron(III) (EDDHSA/Fe) represents one of the promising chelates for the treatment of chlorotic plants. Industrial synthesis of EDDHSA/Fe leads to relevant amounts of o,o-EDDHSA condensation products (o,o-EDDHSAcps) and other secondary products that might have important relevance from the agronomic point of view. However, their chemical structures have remained unknown to date. Analysis of iron complexes by ion-pair reversed-phase chromatography, coupled with electrospray tandem mass spectrometry revealed the presence of the meso-o,o-EDDHSA/Fe, rac-o,o-EDDHSA/Fe, o,p-EDDHSA/Fe regioisomers, the hydroxyl derivative of o,o-EDDHSA/Fe, and the three main EDDHSA condensation products chelating the iron(III) (EDDHSAcps/nFe). However, the chromatographic peaks of EDDHSAcps/Fe are not well resolved due to the large numbers of stereoisomers and the poor efficiency of the ion-pair reversed-phase separation method. An alternative chromatographic method is based on porous graphitic carbon (PGC) separation after pre-column decomplexation of the chelates with trifluoracetic acid, which was developed to allow detection of EDDHSA stereo/regioisomers, EDDHSAcps, and low-molecular-weight by-products. This extensive PGC–HPLC–ESI-MS/MS investigation provides quantitative determination of meso-o,o-EDDHSA, rac-o,o-EDDHSA and o,p-EDDHSA, in addition to characterisation of EDDHSAcps and the low-molecular-weight by-products. PGC separation coupled to a triple quadrupole ESI-MS detector allowed characterisation of free ligands using collision-induced dissociation experiments in positive and negative ionisation mode, providing comparative evaluation of EDDHSAcps in three commercial samples. For detection, the PGC–HPLC–ESI-MS/MS is the best method according to the limit of quantification and limit of detection (picomolar and sub-picomolar detection, respectively) for determination of meso-EDDHSA and rac-o,o-EDDHSA. Synthesis, purification and quantification of o,o-EDDHSA and o,p-EDDHSA by 1H-nuclear magnetic resonance are also reported

ROOT FERRIC CHELATE REDUCTASE IS REGULATED BY IRON AND COPPER IN STRAWBERRY PLANTS

In the present experiment, we studied the interaction between copper (Cu) and iron (Fe) in strawberry plants grown in nutrient solutions containing different concentrations of Fe. Plants grown in the absence of iron (Fe0) had the characteristic symptoms of Fe deficiency, with smaller chlorotic leaves, less biomass, acidification of the nutrient solution, and roots that were smaller and less ramified, while no symptoms of Fe deficiency were observed in plants grown with Fe. A greater amount of Cu was found in roots of chlorotic plants than in those grown with Fe, while plants grown with 20 μM of Fe (Fe20) in the nutrient solution had a greater amount of Fe compared with plants from the other treatments. Chlorotic plants (Fe0) and plants grown with the greatest level of Fe (Fe20) had a greater root ferric chelate reductase (FC-R; EC 1.16.1.17) activity compared with the other treatments with 5 or 10 μM Fe in the nutrient solution. The same pattern was obtained for relative FC-R mRNA concentration and for the sum of Fe and Cu contents in shoots (leaves plus crowns). The DNA obtained from amplification of the FC-R mRNA was cloned and several of the inserts analysed by single strand confirmation polymorphism (SSCP). Although there were different SSCP patterns in the Fe20 treatment, all the inserts that were sequenced were very similar, excluding the hypothesis of more than one FC-R mRNA species being present. The results suggest that Cu as well as Fe is involved in FC-R expression and activity, although the mechanism involved in this regulation is unknown so far. Both small contents of Fe and Cu in plants led to an over-expression of the FC-R gene and enhanced FC-R activity in strawberry roots.

Ribonuclease II preserves chloroplast RNA homeostasis by increasing mRNA decay rates, and cooperates with polynucleotide phosphorylase in 3′ end maturation

Ribonuclease R (RNR1) and polynucleotide phosphorylase (cpPNPase) are the two known 3'→5' exoribonucleases in Arabidopsis chloroplasts, and are involved in several aspects of rRNA and mRNA metabolism. In this work, we show that mutants lacking both RNR1 and cpPNPase exhibit embryo lethality, akin to the non-viability of the analogous double mutant in Escherichia coli. We were successful, however, in combining an rnr1 null mutation with weak pnp mutant alleles, and show that the resulting chlorotic plants display a global reduction in RNA abundance. Such a counterintuitive outcome following the loss of RNA degradation activity suggests a major importance of RNA maturation as a determinant of RNA stability. Detailed analysis of the double mutant demonstrates that the enzymes catalyze a two-step maturation of mRNA 3' ends, with RNR1 polishing 3' termini created by cpPNPase. The bulky quaternary structure of cpPNPase compared with RNR1 could explain this activity split between the two enzymes. In contrast to the double mutants, the rnr1 single mutant overaccumulates most mRNA species when compared with the wild type. The excess mRNAs in rnr1 are often present in non-polysomal fractions, and half-life measurements demonstrate a substantial increase in the stability of most mRNA species tested. Together, our data reveal the cooperative activity of two 3'→5' exoribonucleases in chloroplast mRNA 3' end maturation, and support the hypothesis that RNR1 plays a significant role in the destabilization of mRNAs unprotected by ribosomes.