José-Maria Garcia-Mina

Action of humic acid on promotion of cucumber shoot growth involves nitrate-related changes associated with the root-to-shoot distribution of cytokinins, polyamines and mineral nutrients.

Numerous studies have reported the ability of humic substances to increase shoot growth in different plant species cultivated under diverse growth conditions. However, the mechanism responsible for this effect of humic substances is poorly understood. It is possible that the shoot promoting effect of humic substances involves a primary effect on root H(+)-ATPase activity and nitrate root-shoot distribution that, in turn, causes changes in the root-shoot distribution of certain cytokinins, polyamines and abscisic acid, thus affecting shoot growth. We investigated this hypothesis in the present study. The results showed that the root application of a purified humic acid causes a significant increase in shoot growth that is associated with an enhancement in root H(+)-ATPase activity, an increase in nitrate shoot concentration, and a decrease in roots. These effects were associated with significant increases in the shoot concentration of several cytokinins and polyamines (principally putrescine), concomitant with decreases in roots. Likewise, these changes in the root-shoot distribution of diverse active cytokinins correlated well to significant changes in the root-shoot distribution of several mineral nutrients. These results, taken together, indicate that the beneficial effects of humic substances on shoot development in cucumber could be directly associated with nitrate-related effects on the shoot concentration of several active cytokinins and polyamines (principally putrescine).

Leaf mineral nutrient remobilization during leaf senescence and modulation by nutrient deficiency

Higher plants have to cope with fluctuating mineral resource availability. However, strategies such as stimulation of root growth, increased transporter activities, and nutrient storage and remobilization have been mostly studied for only a few macronutrients. Leaves of cultivated crops (Zea mays, Brassica napus, Pisum sativum, Triticum aestivum, Hordeum vulgare) and tree species (Quercus robur, Populus nigra, Alnus glutinosa) grown under field conditions were harvested regularly during their life span and analyzed to evaluate the net mobilization of 13 nutrients during leaf senescence. While N was remobilized in all plant species with different efficiencies ranging from 40% (maize) to 90% (wheat), other macronutrients (K–P–S–Mg) were mobilized in most species. Ca and Mn, usually considered as having low phloem mobility were remobilized from leaves in wheat and barley. Leaf content of Cu–Mo–Ni–B–Fe–Zn decreased in some species, as a result of remobilization. Overall, wheat, barley and oak appeared to be the most efficient at remobilization while poplar and maize were the least efficient. Further experiments were performed with rapeseed plants subjected to individual nutrient deficiencies. Compared to field conditions, remobilization from leaves was similar (N–S–Cu) or increased by nutrient deficiency (K–P–Mg) while nutrient deficiency had no effect on Mo–Zn–B–Ca–Mn, which seemed to be non-mobile during leaf senescence under field conditions. However, Ca and Mn were largely mobilized from roots (-97 and -86% of their initial root contents, respectively) to shoots. Differences in remobilization between species and between nutrients are then discussed in relation to a range of putative mechanisms.

Microarray analysis of humic acid effects on Brassica napus growth: Involvement of N, C and S metabolisms

Background & aimsWinter rapeseed (Brassica napus) is characterized by a low N recovery in seeds and requires high rates of fertilization to maintain yield. Its nutrient use efficiency could be improved by addition of a biostimulant such as humic acids whose physiological effects have been described previously in some plant species. However, to our knowledge, no study has focused on transcriptomic analyses to determine metabolic targets of this extract.MethodsA preliminary screening of ten humic acids revealed a significant effect of one of them (HA7) on rapeseed root growth. Microarray analysis was then used on HA7-treated or non-treated plants to characterize changes in gene expression that were further supported by physiological evidence.ResultsStimulation of nitrogen uptake (+15% in shoots and +108% in roots) and assimilation was found to be increased in a similar manner to growth while sulfate content (+76% in shoots and +137% in roots) was more strongly stimulated leading to higher sulfate accumulation. In parallel, microscopic analysis showed an enhancement of chloroplast number per cell.ConclusionIt is therefore suggested that HA7, which promotes plant growth and nutrient uptake, could be used as a supplementary tool to improve rapeseed nitrogen use efficiency.

The importance of nitrate in ameliorating the effects of ammonium and urea nutrition on plant development: the relationships with free polyamines and plant proline contents

In order to investigate the possible involvement of free polyamines and proline in the mechanism underlying the action of nitrate in correcting the negative effects associated with ammonium and urea nutrition in certain plant species, we studied plant contents of free polyamines and proline associated with nitrogen nutrition involving different nitrogen forms (nitrate, ammonium, urea) in two plant species, wheat and pepper. The results showed that ammonium nutrition and, to a lesser extent, urea nutrition were associated with significant increases in plant putrescine content that were well correlated with reductions in plant growth. These negative effects of ammonium and urea nutrition were corrected by the presence of nitrate in the nutrient solution; the presence of nitrate was also related to a significant decrease in the plant putrescine content. These results are compatible with a specific effect of nitrate reducing ammonium accumulation through the improvement of ammonium assimilation. As for the plant proline content, in pepper a slight increase in this parameter was associated with ammonium and urea nutrition, but it was also decreased by the presence of nitrate in the nutrient solution. These changes, however, were not so clearly related to the variations in plant growth as in the case of putrescine content. These results are compatible with the hypothesis that putrescine biosynthesis might be related to proline degradation by a specific pathway related to ammonium detoxification.

Interaction of Different Iron Chelates with an Alkaline and Calcareous Soil: A Complementary Methodology to Evaluate the Performance of Iron Compounds in the Correction of Iron Chlorosis

Abstract A great number of studies have shown that the stability of iron chelates as a function of pH is not the unique parameter that must be considered in order to evaluate the potential effectiveness of Fe‐chelates to correct iron chlorosis in plants cultivated in alkaline and calcareous soils. In fact, other factors, such as soil sorption on soil components or the competition among Fe and other metallic cations for the chelating agent in soil solution, have a considerable influence on the capacity of iron chelates to maintain iron in soil solution available to plants. In this context, the aim of this work is to study the variation in concentration of the main iron chelates employed by farmers under field conditions—Fe‐EDDHA (HA), Fe‐EDDHMA (MA), Fe‐EDDHSA (SA), Fe‐EDDCHA (CA), Fe‐EDTA (EDTA), and Fe‐DTPA (DTPA)—in the soil solution of a calcareous soil over time. To this end, soil incubations were carried out using a soil:Fe solution ratio corresponding to soil field capacity, at a temperature of 23°C. The soil used in the experiments was a calcareous soil with a very low organic matter content. The variation in concentration of Fe and Fe‐chelates in soil solution over time were obtained by measuring the evolution in soil solution of both the concentration of total Fe (measured by AAS), and the concentration of the ortho‐ortho isomers for Fe‐EDDHA and analogs or chelated Fe for Fe‐EDTA and Fe‐DTPA (measured by HPLC). The following chelate samples were used: a HA standard prepared in the laboratory and samples of HA, MA, SA, CA, Fe‐EDTA, and Fe‐DTPA obtained from commercial formulations present in the market. The percentage of iron chelated as ortho‐ortho isomers for HAs was: HA standard (100%); HA (51.78%); MA (60.06%); SA (22.50%); and CA (27.28%). In the case of Fe‐EDTA and Fe‐DTPA the percentages of chelated iron were 96.09 and 99.12, respectively. Results show that it is possible to classify the potential effectiveness of the different types of iron chelates used in our experiments as a function of two practical approaches: (i) considering the variation of total iron in soil solution over time, MA is the best performing product, followed by HA, CA, SA, DTPA, EDTA, and ferrous sulfate in the order listed and (ii) considering the capacity of the different iron chelates to maintain the fraction of chelated iron (ortho‐ortho isomers for HA, MA, SA, and CA and total chelated iron for EDTA and DTPA) in soil solution, the order is: SA > CA > HA > MA > EDTA ≈ DTPA. This result, that is related to the nature of the chelate and does not depend on the degree of chelated Fe in the products, indicates that SA and CA might be very efficient products to correct iron chlorosis. Finally, our results also indicate the suitability of this soil incubation methodology to evaluate the potential efficiency of iron compounds to correct iron chlorosis.

Hydroponics versus field lysimeter studies of urea, ammonium and nitrate uptake by oilseed rape(Brassica napus L.)

N-fertilizer use efficiencies are affected by their chemical composition and suffer from potential N-losses by volatilization. In a field lysimeter experiment, (15)N-labelled fertilizers were used to follow N uptake by Brassica napus L. and assess N-losses by volatilization. Use of urea with NBPT (urease inhibitor) showed the best efficiency with the lowest N losses (8% of N applied compared with 25% with urea alone). Plants receiving ammonium sulphate, had similar yield achieved through a better N mobilization from vegetative tissues to the seeds, despite a lower N uptake resulting from a higher volatilization (43% of applied N). Amounts of (15)N in the plant were also higher when plants were fertilized with ammonium nitrate but N-losses reached 23% of applied N. In parallel, hydroponic experiments showed a deleterious effect of ammonium and urea on the growth of oilseed rape. This was alleviated by the nitrate supply, which was preferentially taken up. B. napus was also characterized by a very low potential for urea uptake. BnDUR3 and BnAMT1, encoding urea and ammonium transporters, were up-regulated by urea, suggesting that urea-grown plants suffered from nitrogen deficiency. The results also suggested a role for nitrate as a signal for the expression of BnDUR3, in addition to its role as a major nutrient. Overall, the results of the hydroponic study showed that urea itself does not contribute significantly to the N nutrition of oilseed rape. Moreover, it may contribute indirectly since a better use efficiency for urea fertilizer, which was further increased by the application of a urease inhibitor, was observed in the lysimeter study.

Copper-Deficiency in Brassica napus Induces Copper Remobilization, Molybdenum Accumulation and Modification of the Expression of Chloroplastic Proteins

During the last 40 years, crop breeding has strongly increased yields but has had adverse effects on the content of micronutrients, such as Fe, Mg, Zn and Cu, in edible products despite their sufficient supply in most soils. This suggests that micronutrient remobilization to edible tissues has been negatively selected. As a consequence, the aim of this work was to quantify the remobilization of Cu in leaves of Brassica napus L. during Cu deficiency and to identify the main metabolic processes that were affected so that improvements can be achieved in the future. While Cu deficiency reduced oilseed rape growth by less than 19% compared to control plants, Cu content in old leaves decreased by 61.4%, thus demonstrating a remobilization process between leaves. Cu deficiency also triggered an increase in Cu transporter expression in roots (COPT2) and leaves (HMA1), and more surprisingly, the induction of the MOT1 gene encoding a molybdenum transporter associated with a strong increase in molybdenum (Mo) uptake. Proteomic analysis of leaves revealed 33 proteins differentially regulated by Cu deficiency, among which more than half were located in chloroplasts. Eleven differentially expressed proteins are known to require Cu for their synthesis and/or activity. Enzymes that were located directly upstream or downstream of Cu-dependent enzymes were also differentially expressed. The overall results are then discussed in relation to remobilization of Cu, the interaction between Mo and Cu that occurs through the synthesis pathway of Mo cofactor, and finally their putative regulation within the Calvin cycle and the chloroplastic electron transport chain.

Two Biostimulants Derived from Algae or Humic Acid Induce Similar Responses in the Mineral Content and Gene Expression of Winter Oilseed Rape (Brassica napus L.)

Different strategies, known as crop biofortification, can be used to increase micronutrient concentrations in harvested parts to reduce nutrient deficiencies in the human diet. Apart from fertilization and genetic selection, a more environmentally friendly, less expensive, and more immediate solution could rely on the use of biostimulants derived from natural materials. Two biostimulants, AZAL5 and HA7, which are derived from seaweed and black peat, respectively, have been previously described as promoting growth of Brassica napus and having a substantial effect on gene expression. They were further studied to evaluate their effects on N and S and a wide range of other nutrients (that is, K, Ca, P, Mg, Fe, Na, Mn, B, Si, Cu, and Zn). Providing these two biostimulants in the nutrient solution did not change the mineral supply significantly, but they mostly stimulated root growth and macronutrient uptake (N, S, K, and P) at a level similar to growth. Both biostimulants also stimulate chloroplast division. More surprisingly, they also increased Mg, Mn, Na, and Cu plant concentrations and root-to-shoot translocation of Fe and Zn. These observations were associated with an increased expression of a Cu transporter (COPT2) and NRAMP3, a gene putatively involved in Fe and Zn translocation. Overall, this study showed that specific nutrient balance and transport were stimulated by both biostimulants more significantly than growth, offering new perspectives for biofortification strategies.

A physiological and molecular study of the effects of nickel deficiency and phenylphosphorodiamidate (PPD) application on urea metabolism in oilseed rape ( Brassica napus L.)

Background and aimsUrea is the major nitrogen (N) form supplied as fertilizer in agriculture. However, urease, a nickel-dependent enzyme, allows plants to use external or internally generated urea as a nitrogen source. Since a urease inhibitor is frequently applied in conjunction with urea fertilizer, the N-metabolism of plants may be affected. The aim of this study was to determine physiological and molecular effects of nickel deficiency and a urease inhibitor on urea uptake and assimilation in oilseed rape.MethodsPlants were grown on hydroponic solution with urea as the sole N source under three treatments: plants treated with nickel (+Ni) as a control, without nickel (−Ni) and with nickel and phenylphosphorodiamidate (+Ni+PPD). Urea transport and assimilation were investigated.ResultsThe results show that Ni-deficiency or PPD supply led to reduced growth and reduced 15N-uptake from urea. This effect was more pronounced in PPD-treated plants, which accumulated high amounts of urea and ammonium. Thus, Ni-deficiency or addition of PPD, limit the availability of N and decreased shoot and root amino acid content. The up-regulation of BnDUR3 in roots indicated that this gene is a component of the stress response to nitrogen-deficiency. A general decline of glutamine synthetase (GS) activity and activation of glutamate dehydrogenase (GDH) and increases in its expression level were observed in control plants. At the same time, in (−N) or (+Ni+PPD) treated plants, no increases in GS or GDH activities and expression level were found.ConclusionsOverall results showed that plants require Ni as a nutrient (while most widely used nutrient solutions are devoid of Ni), whether they are grown with or without a urea supply, and that urease inhibitors may have deleterious effects at least in hydroponic grown oilseed rape.

Distribution and Secondary Effects of EDDHA in Some Vegetable Species

Abstract Tomato (Lycopersicum esculentum), cucumber (Cucumis sativus), pepper (Capsicum annuum), and lettuce (Lactuca sativa) were grown on rockwool or perlite substrate with nutrient solution. Fe was administered as the Fe complex of the chelator ethylenediamine di-(o-hydroxyphenylacetic acid) (EDDHA) or Fe(NH4)2(SO4)2 in the nutrient solution or as inorganic iron in the substrate. Roots and leaves of plants grown on Fe-EDDHA contained EDDHA in quantities up to 0.27 × the amount of Fe, which is interpreted as an indication of the contribution of passive chelate absorption to Fe uptake. Fruits of tomato and pepper, and leaves of lettuce contained only traces of EDDHA. Breakdown of the chelator in leaves of pepper and tomato is estimated to have been between 0.5 and 2% per day. In tomato fruits, lycopene content was lowered in plants growing on Fe-EDDHA. Cucumber growing on Fe-EDDHA suffered from serious infection by the mildew Sphaerotheca fusca; the plants growing on an inorganic source of iron were resistant. These results exemplify physiological effects of EDDHA other than those directly associated with iron nutrition.