Combat of iron-deprivation through a plant growth promoting fluorescent Pseudomonas strain GRP3A in mung bean (Vigna radiata L. Wilzeck)

Microorganisms and plants sustain themselves under iron-deprived conditions by releasing siderophores. Among others, fluorescent pseudomonads are known to exert extensive biocontrol action against soil and root borne phytopathogens through release of antimicrobials and siderophores. In this study, production and regulation of siderophores by fluorescent Pseudomonas strain GRP3A was studied. Among various media tested, standard succinate medium (SSM) promoted maximum siderophore production of 56.59 mg l(-1). There were low levels of siderophore in complex media like King's B medium, trypticase soya medium and nutrient medium (41.27, 29.86 and 27.63 mg l(-1)), respectively. In defferrated SSM, siderophore level was quantified to be 68.74 mg l(-1). Supplementation with iron (FeCl3) resulted in decreased siderophore levels depending on concentration. Siderophore production was promoted by Zn2+ (78.94 mg l(-1)), Cu2+ (68.80 mg l(-1)) whereas Co2+ (57.33 mg l(-1)) and Fe3+ reduced siderophore production (37.44 mg l(-1) as compared to control (55.97 mg l(-1)). Strain GRP3A showed plant growth promotion under iron limited conditions.     

Impact of iron deficiency and iron re-supply during the early stages of vegetative development in maize (Zea mays L.)

The consequences of iron deficiency and iron re-supply were evaluated during the early stages of growth and development of young maize plantlets grown hydroponically in the absence of iron. Various parameters, such as fresh and dry weights, and the concentration of chlorophylls, iron, copper, manganese, calcium, magnesium and potassium in leaves, were measured at various times during the first 15 d of culture. Ten-day-old maize plantlets grown without iron displayed severe alterations, with a 50% decrease in iron and chlorophyll concentrations in leaves, and serious impairments in mitochondria and chloroplast ultrastructure. In contrast, neither leaf nor root growth, nor other mineral concentrations other than iron were significantly affected at this stage of development. In an attempt to characterize proteins potentially involved in iron nutrition or the adaptative response to iron starvation, comparative 2D-gel electrophoretic analysis of polypeptides was carried out on soluble and membrane fractions prepared from leaves and roots of iron-deficient and iron-sufficient 10-d-old maize plantlets. Two polypeptides (11 and 17 kDa, pI of about 6.8) from the microsomal fraction of leaves were found to be repressed under iron-deficient conditions. Some other polypeptides were found to he induced in microsomal fractions either from roots or leaves. Significant variations in the concentration of most of these polypeptides were observed from one experiment to another. It can be concluded from this study that, at this early stage of maize vegetative growth and development, molecular variations induced by iron deficiency do not affect major house-keeping proteins, but probably affect very specific events depending on low abundance proteins.     

Ameliorative role of zinc on maize growth (Zea mays L.) under salt-affected soil conditions

A greenhouse experiment, growing maize for six weeks, was conducted to evaluate the ameliorative role of Zn (0 and 10 ppm Zn) under saline (ECe4, 8 and 12 mmhos/cm), Sodic (ESP 10, 20 and 30) and saline-sodic (all possible combinations of above salinity and sodicity levels), and normal soil conditions using a sandy loam (Typic Ustochrepts) soil sample.Zinc ameliorated plant growth under salt-affected soil conditions. Ameliorative effect was more under sodic than under saline or saline-sodic soil conditions. Shoot yield decreased with Salinity level of 12 mmhos/cm, and ESP 30 and adverse effects were accentuated with increasing level of ESP and Salinity, respectively.Shoot Zn increased with applied Zn. Increasing sodicity in soil under Zn deficient or low salinity conditions generally decreased shoot Zn, whereas the low level of soil salinization counteracted the adverse effect of high sodicity. Shoot Na increased but K decreased with increasing sodicity and salinity in soil. Shoot Na decreased but K increased with applied Zn. Shoot Ca increased with salinity levels of 4 and 8 mmhos/cm, but decreased with 12 mmhos/cm at 0 Zn level. Sodicity decreased shoot Ca, whereas Zn counteracted adverse effect of high sodicity. Shoot Mg generally increased with increasing salinity, but decreased with increasing sodicity. Zinc had no definite effect. Shoot Ca/Na and K/Na ratios were widened with Zn and narrowed down with high ESP.The effects of salinity, sodicity, and Zn on plant growth and its composition were generally associated with their respective roles in dry matter production, and inter-ionic relationships among Ca, Mg, K, Na and Zn in soils and plants.Contribution from the Department of Soils, Haryana Agricultural University, Hissar, 125004, Indiaformer Research Fellow, respectively.     

Improving the mineral reserves and protein quality of maize (Zea mays L.) kernels using unique genes

The effects of the maize genes, o ₂ and Mal, on the concentrations of mineral nutrient cations and amino acid levels in mature maize (Zea mays L) kernels of various inbred lines were studied. Previously, the o ₂ gene has been used to improve the protein quality and increase the mineral nutrient content of kernels from some inbred lines. Genotypes possessing the Mal (multiple aleurone layer) gene, contain more than one row of aleurone cells in their kernels and this gene enhances the effect of the o ₂gene on improving kernel protein quality. Incorporating these genes into the maize genome increased accumulation of several mineral nutrients (including Ca, Mg, Zn, Fe, Mn, Zn and Cu) in some of the experimental lines studied. The physiological basis for this increase of mineral nutrients in the kernels is discussed. The effect of the Mal gene on the kernel amino acid composition and protein quality was also examined. Possibly, these genes could be used in combination in breeding programs to improve kernel quality and nutritional value of maize.     

Thiols in cadmium- and copper-treated maize (Zea mays L.)

Maize plants (Zea mays L. cv. Honeycomb F-1) were grown on quartz sand containing amounts of Cd or Cu which resulted in comparable internal contents in the roots. Fresh and dry weights and the content of Cd or Cu were measured in roots and shoots after eight weeks. In addition, cysteine, γ-glutamylcysteine (γEC), glutathione (GSH) and the thiols in heavy-metal-binding peptides (HMBPs) were determined in the roots. At low internal contents, Cd and Cu inhibited root growth to the same extent. Inhibition by Cu was enhanced, however, at high internal contents, indicating that Cu was more toxic than Cd. Separation of extracts from roots of Cd- and Cutreated plants on a Sephadex G-50 column resulted in HMBP complexes with relative molecular masses (M_rs) of 6200 and 7300, respectively. Separation of these HMBP-complexes using HPLC resulted in a distinct pattern of thiol compounds for each heavy metal. The accumulation of HMBPs was linearly dependent on the content of Cd at all values examined. In Cu-treated roots, HMBP accumulation was linearly dependent on the internal Cu content only up to 7.1 μmol·g^?1 dry weight. At internal contents which caused an enhanced inhibition of root growth, no further significant increase in the HMBP content was detected. At these internal Cu contents an increased transport of Cu to the shoot was measured. This result indicates that HMBPs are involved in reducing heavy-metal transport from roots to shoots.     

Effect of plant growth promoting rhizobacteria containing ACC-deaminase on maize (Zea mays L.) growth under axenic conditions and on nodulation in mung bean (Vigna radiata L.)

AIMS: This study was conducted to test the hypothesis that the bacterial strains possessing 1-aminocyclopropane-1-carboxylic acid (ACC)-deaminase activity may also promote growth of inoculated plants and could increase nodulation in legumes upon co-inoculation with rhizobia. METHODS AND RESULTS: Several rhizobacteria were isolated from maize rhizosphere through enrichment on ACC as a sole N source. Purified isolates were screened for growth promotion in maize under axenic conditions and for in vitro ACC-deaminase activity. A significant positive correlation was observed between in vitro ACC-deaminase activity of bacterial cells and root elongation. None of the isolates produced auxins. Bradyrhizobium japonicum produced less amount of auxins but did not carry ACC-deaminase activity. Results of pot experiment revealed that co-inoculation with Bradyrhizobium and plant growth promoting rhizobacteria (PGPR) isolates enhanced the nodulation in mung bean compared with inoculation with Bradyrhizobium alone. CONCLUSIONS: It is highly expected that inoculation with rhizobacteria containing ACC-deaminase hydrolysed endogenous ACC into ammonia and alpha-ketobutyrate instead of ethylene. Consequently, root and shoot growth as well as nodulation were promoted. SIGNIFICANCE AND IMPACT OF THE STUDY: The ACC-deaminase trait could be employed as an efficient tool to screen effective PGPR, which could be successfully used as biofertilizers to increase the growth of inoculated plants as well as nodulation in legumes.     

Effects of high temperature on the germination of maize (Zea mays L.)

Poor emergence of maize seedlings, due to high soil temperatures, is a major limitation of crop potential in the lowland tropics. Ability to germinate at high temperature (>c. 37° C) is related to the temperature sensitivity of the embryo, and there is considerable genotypic variation for this character.Respiration and mitochondrial phosphorylation proceed normally in seeds imbibing at 41° C, and ATP levels are adequate for germination. However, the specific activities of several important enzymes are lower, and the rate of protein synthesis is severely reduced compared with seeds imbibing at 28° C. The depression of the rate of protein synthesis in the embryos of several tropical hybrids imbibing at high temperature correlated with their known temperature sensitivity. It is concluded that protein synthesis is an especially temperature sensitive process in germinating maize embryos, and that this is the principal reason for the sensitivity of germinating maize seeds to high temperature.Abbreviations ADP adenosine-5-diphosphate - ATP adenosine-5-triphosphate - BSA bovine serum albumin - EDTA ethylenediaminetetra-acetic acid - HEPES N-2-hydroxyethylpiperazinc-N-2-ethanesulphonic acid - NADH nicotinamide-adenine dinucleotide, reduced form - PPO 2, 5-diphenyloxazole - PVP polyvinylpyrrolidone - SEM standard error of the mean - tris tris (hydroxymethyl)-methylamine     

Associative bacteria influence maize (Zea mays L.) growth,physiology and root anatomy under different nitrogen levels

• Despite the great diversity of plant growth‐promoting bacteria (PGPB ) with potential to partially replace the use of N fertilisers in agriculture, few PGPB have been explored for the production of commercial inoculants, reinforcing the importance of identifying positive plant–bacteria interactions. Aiming to better understand the influence of PGPB inoculation in plant development, two PGPB species with distant phylogenetic relationship were inoculated in maize.
• Maize seeds were inoculated with Bacillus sp. or Azospirillum brasilense . After germination, the plants were subjected to two N treatments: full (N+) and limiting (N?) N supply. Then, anatomical, biometric and physiological analyses were performed.
• Both PGPB species modified the anatomical pattern of roots, as verified by the higher metaxylem vessel element (MVE ) number. Bacillus sp. also increased the MVE area in maize roots. Under N+ conditions, both PGPB decreased leaf protein content and led to development of shorter roots; however, Bacillus sp. increased root and shoot dry weight, whereas A. brasilense increased photosynthesis rate and leaf nitrate content. In plants subjected to N limitation (N?), photosynthesis rate and photosystem II efficiency increased in maize inoculated with Bacillus sp., whilst A. brasilense contained higher ammonium, amino acids and total soluble sugars in leaves, compared to the control.
• Plant developmental and metabolical patterns were switched by the inoculation, regardless of the inoculant bacterium used, producing similar as well as distinct modifications to the parameters studied. These results indicate that even non‐diazotrophic inoculant strains can improve the plant N status as result of the morpho‐anatomical and physiological modifications produced by the PGPB .

     

Herbicidal influence on the uptake of phosphorus by paddy (Oryza sativa L.) and maize (Zea mays L.)

Summary Phosphorus uptake is significantly reduced in paddy and maize by the application of atrazine, DPA, EPTC and 2, 4-D from quarter normal to twice normal levels except 2 kg/ha of 2, 4-D which increased phosphorus absorption in maize. The most effective treatments are 2 kg/ha atrazine and 8 kg/ha EPTC which reduced P uptake to 55.50 and 48.12 % of control in paddy and maize respectively. Absorption of³²P by paddy and maize has also been reduced significantly by the application of above mentioned herbicides with the exception of 1 kg/ha EPTC which slightly increased it in maize.     

A low-pressure gene gun for genetic transformation of maize (Zea mays L.)

We have successfully used the low-pressure BioWare gene gun, developed for gene transfer in animal cells, for plant tissues. The BioWare device is easy to manipulate. Just 50 psi helium pressure was sufficient to transfer foreign genes into the aleurone layer and embryo of maize without causing tissue damage in the impact area. As shown by expression signals from invasive histochemical β-glucuronidase (GUS) activity, the foreign reporter gene expressed well in bombarded tissues. This successful GUS-transient expression extends the application of this low-pressure gene gun from animal cells to plant tissues.     

Dehydrogenase and urease activities of maize (Zea mays L.) field soils

Summary Dehydrogenase and urease activities, bacterial and fungal populations and physicochemical characteristics of maize (Zea mays L.) field soils have been studied for one crop cycle. A comparison has been made among soils of three different agricultural systemsviz permanent agriculture on plain lands in valleys, recently introduced terrace land agriculture and age old ‘slash and burn’ type of shifting agriculture on slopes. Results demonstrate that the enzyme activities, microbial population as well as most of the physico-chemical characteristics of soils followed the trend permanent agriculture on plain lands>terrace land agriculture>‘slash and burn’ type of shifting agriculture. Moisture and nutrient levels and topography of the lands were found to be major factors responsible for the trend.     

Subcellular localization of glutelin-2 in maize (Zea mays L.) endosperm

Accumulation of the 28 KD protein of the glutelin-(G2) fraction was followed in developing maize endosperm, using sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and peak integration of scanned gels. 28 KD glutelin-2 could already be observed from 15 days after pollination and its accumulates reached a plateau during the second half of the development period. The process of biosynthesis of 28 KD glutelin-2 and zeins occurs in a parallel way. Subcellular fractions obtained from linear sucrose gradient centrifugation of developing maize endosperms were analyzed by SDS-PAGE and immunoblotting using a serum reacting against glutelin-2 and 14 KD Z2. Glutelin-2 was found to be present in the protein bodies when subcellular fractionation was carried out without dithiothreitol (DTT). The presence of a reducing agent causes the elution of glutelin-2 from protein bodies. Immunocytochemical labelling using the protein A-colloidal gold technique in protein bodies incubated with anti-G2 IgG revealed that G2 is located mainly in the periphery of protein bodies. These results are interpreted as indicating a structural role for glutelins in protein bodies.     

Mapping of quantitative trait loci associated with chilling tolerance in maize (Zea mays L.) seedlings grown under field conditions

The effect of low growth temperature on morpho-physiological traits of maize was investigated by the means of a QTL analysis in a segregating F(2:3) population grown under field conditions in Switzerland. Chlorophyll fluorescence parameters, leaf greenness, leaf area, shoot dry weight, and shoot nitrogen content were investigated at the seedling stage for two years. Maize was sown on two dates in each year; thus, plants sown early were exposed to low temperature, whereas those sown later developed under more favourable conditions. The main QTLs involved in the functioning of the photosynthetic apparatus at low temperature were stable across the cold environments and were also identified under controlled conditions with suboptimal temperature in a previous study. Based on the QTL analysis, relationships between chlorophyll fluorescence parameters and leaf greenness were moderate. This indicates that the extent and functioning of the photosynthetic machinery may be under different genetic control. The functioning of the photosynthetic apparatus in plants developed at low temperature in the field did not noticeably affect biomass accumulation; since there were no co-locations between QTLs for leaf area and shoot dry weight, biomass accumulation did not seem to be carbon-limited at the seedling stage under cool conditions in the field.     

Proteomic analysis of shoot-borne root initiation in maize (Zea mays L.)

Postembryonically formed shoot-borne roots make up the major backbone of the adult maize root stock. In this study the abundant soluble proteins of the first node (coleoptilar node) of wild-type and mutant rtcs seedlings, which do not initiate crown roots, were compared at two early stages of crown root formation. In Coomassie Bluestained 2-D gels, representing soluble proteins of coleoptilar nodes 5 and 10 days after germination, 146 and 203 proteins were detected, respectively. Five differentially accumulated proteins (> two-fold change; t-test: 95% significance) were identified in 5-day-old and 14 differentially accumulated proteins in 10-day-old coleoptilar nodes of wild-type versus rtcs. All 19 differentially accumulated proteins were identified via ESI MS/MS mass spectrometry. Five differentially accumulated proteins, including a regulatory G-protein and a putative auxin-binding protein, were further analyzed at the RNA expression level. These experiments confirmed differential gene expression and revealed subtle developmental regulation of these genes during early coleoptilar node development. This study represents the first proteomic analysis of shoot-borne root initiation in cereals and will contribute to a better understanding of the molecular basis of this developmental process unique to cereals.     

Genomic prediction of seedling root length in maize (Zea mays L.)

Genotypes with extreme phenotypes are valuable for studying ‘difficult’ quantitative traits. Genomic prediction (GP) might allow the identification of such extremes by phenotyping a training population of limited size and predicting genotypes with extreme phenotypes in large sequences of germplasm collections. We tested this approach employing seedling root traits in maize and the extensively genotyped Ames Panel. A training population made up of 384 inbred lines from the Ames Panel was phenotyped by extracting root traits from images using the software program aria . A ridge regression best linear unbiased prediction strategy was used to train a GP model. Genomic estimated breeding values for the trait ‘total root length’ (TRL) were predicted for 2431 inbred lines, which had previously been genotyped by sequencing. Selections were made for 100 extreme TRL lines and those with the predicted longest or shortest TRL were validated for TRL and other root traits. The two predicted extreme groups with regard to TRL were significantly different (P = 0.0001). The difference in predicted means for TRL between groups was 145.1 cm and 118.7 cm for observed means, which were significantly different (P = 0.001). The accuracy of predicting the rank between 1 and 200 of the validation population based on TRL (longest to shortest) was determined using a Spearman correlation to be ρ = 0.55. Taken together, our results support the idea that GP may be a useful approach for identifying the most informative genotypes in sequenced germplasm collections to facilitate experiments for quantitative inherited traits.     

Intron-mediated enhancement of gene expression in maize (Zea mays L.) and bluegrass (Poa pratensis L.)

We report a strength comparison of a large variety of monocot and dicot intron-containing fragments inserted in the 5 untranslated leader, between the CaMV 35S promoter and the uidA gene (coding for the ß-glucuronidase: GUS). Relative strengths of the intron-containing fragments were evaluated by comparing transient GUS expression after particle bombardment in embryogenic maize and bluegrass suspension cultures. Our results confirm a dramatic dependence on the presence of an intron for chimeric gene expression in both species. On average, the maize first intron of ubi1 provided the highest enhancement of gene expression in maize and bluegrass (71- and 26-fold enhancement, respectively). Half of the introns tested affected gene expression differently in bluegrass and maize. This suggests that the intron-mediated enhancement of gene expression generally obtained with maize may not be fully applicable to all monocots. We also report enhancement of gene expression (92-fold) in a monocot species by a dicot intron (chsA intron).     

A comparison of methods for direct gene transfer into maize (Zea mays L.)

Summary Techniques for transforming intact tissues of cereals were evaluated for their efficacy in transforming immature embryos and Type II callus of maize (Zea mays L.). The techniques used were particle bombardment, tissue electroporation, tissue electrophoresis, and silicon carbide fibers. Each method was assessed in terms of transient β-glucuronidase (GUS) expression. High levels of GUS expression were observed in A188 Type II callus using both tissue electroporation and particle bombardment, with means of 417.8 and 954.5 blue expression units (beu) per g fresh weight (FW) callus, respectively. Only particle bombardment resulted in high transient gene expression in immature embryos, with a mean transformation frequency of 34.8 b.e.u. per embryo. Very low levels of GUS expression were achieved with silicon carbide-mediated gene transfer, even when employing tissues used in the original publication (Black Mexican Sweet suspension cells). GUS expression was not obtained following tissue electrophoretic gene delivery.     

Regulation of flower development in cultured ears of maize (Zea mays L.)

Summary Young ears of maize were cultured in two different liquid media containing either kinetin (KN) or kinetin + gibberellic acid (KN + GA₃) in order to manipulate stamen and gynoecium development. In KN medium, stamens developed and gynoecia aborted in the flowers of the cultured immature ears. In the KN + GA₃ medium, however, ovaries with silks developed and stamens aborted. These differential morphological events were recorded with SEM photomicrographs at regular intervals after excision of ear inflorescences. In addition, the mitotic activity in the developing or aborting organs was determined over a 75-h period. It increased from 6% to 14% in developing organs (i.e. stamens in KN medium, and gynoecia in KN + GA₃ medium) and gradually decreased to 1% in the degenerating organs (i.e. gynoecia in KN medium, and stamens in KN + GA₃ medium) by 45 h of culture. The mitotic activity reached zero in degenerating flower organs by 75 h of culture. Whether these differential sensitivities to the exogenously applied members of these two plant growth regulator classes are unique to our in vitro system or reflect a more general control feature of in vivo inflorescences must await further clarification.