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Phosphorus is an element with limited availability in the soil. For this reason, to supply phosphorus to crops, farmers depend on chemical fertilization, which affects the production costs and the environmental quality of the soil and water. However, this situation can be mitigated by using phosphorus-solubilizing fungi. Therefore, the objectives of this work were to identify seven dematiaceous endophytic fungi (Dark Septate Endophytes (DSE) and non-DSE), isolated from roots of a commercial barley crop, and to evaluate their characteristics and behavior in vitro regarding parameters that allow their description and potential use to make fertilizer use more efficient and thus reduce the amount of fertilizer required. The growth rate of each strain at different temperatures and pH values, and their ability to solubilize iron (FePO4), aluminum (AlPO4) and calcium (Ca3(PO4)2) phosphates were evaluated. The fungal isolates belonged to four orders: Pleosporales, Xylariales, Hypocreales and Chaetothyriales. The strains had their highest growth rate between 16°C and 30°C, and a great diversity of responses to the different pH values evaluated. Furthermore, all strains showed ability to solubilize Ca3(PO4)2, two to solubilize AlPO4, and one to solubilize FePO4. The diversity observed among the fungal isolates in terms of their response to pH, temperature, and media containing different insoluble phosphates suggests their adaptability to a wide range of environments, and makes them suitable for further studies aimed to determine their potentiality as biofertilizers.

Context In the malting industry, the low protein content of barley grains is a major issue. A useful strategy to overcome this problem is to complement the initial fertilisation with a foliar application of nitrogen (N) near anthesis. Aims This study aimed to advance knowledge of the metabolic adjustments displayed by plants grown under common management practices in response to foliar N fertilisation at anthesis. Methods Field experiments with two different rates of foliar N application near anthesis were performed at three commercial sites under the common practice of each farmer. Key results Grain protein content increased upon foliar fertilisation in all sites without affecting leaf senescence or N remobilisation efficiency. Barley plants also showed a rapid assimilation of the N applied at anthesis, increasing the leaf N organic pools and the global phloem amino acid mobilisation during grain filling. These results could be attributed to the increase in the gene expression of glutamine synthetase 1 and two amino acid transporters (HvAAP6 and HvAAP7). Conclusions Foliar N applied was rapidly assimilated and exported with high efficiency to the grain with no negative impact over the contribution of N from vegetative organs. Furthermore, phloem N contribution was shown to be of major importance for grain protein content. Implications Foliar fertilisation near anthesis is an efficient strategy to correct grain protein content to meet maltsters’ requirements because it did not affect pre-assimilated N remobilisation, and was useful under the different nutrient availabilities explored here (N sufficiency, N deficiency and sulfur deficiency).

Background and Aims Proteases are responsible for protein degradation during leaf senescence, allowing nutrients to be redirected to sink tissues. In a previous work, we reported that sulphur deficiency produced a delay in the leaf senescence of barley (Hordeum vulgare L.) plants, at both vegetative and reproductive stages. In this work, we analyse the effect of sulphur deficiency on the expression of several genes coding for proteases of different catalytic groups, which have been strongly associated with leaf senescence. Methods Four independent experiments were performed in order to impose low sulphur availability conditions: one of steady-state sulphur deficiency during the vegetative stage and three of sulphur starvation during vegetative and reproductive stages. Key Results Sulphur deficiency inhibited or reduced the senescence-associated induction of seven of the eight proteases analysed. Their induction, as well as senescence and phloem amino acid remobilization, could be achieved with senescence inducers such as methyl-jasmonate (a hormonal stimulus) and darkness, but with different rates of induction dependent on each gene. Sulphur deficiency also exerted an opposite effect on the expression of two cysteine-protease genes (HvSAG12 and HvLEGU) as well as on one serine-protease gene (HvSUBT) according to leaf age and plant phenological stages. All three genes were induced in green leaves but were repressed in senescent leaves of sulphur-deficient plants at the vegetative stage. At the reproductive stage, both cysteine-proteases were only repressed in senescent leaves, while the serine-protease was induced in green and senescent leaves by sulphur deficiency. Conclusions Our results highlight the relevance of adequate sulphur nutrition in order to ensure leaf senescence onset and induction of protease genes, which will consequently impact on grain protein composition and quality. In addition, our results provide evidence that leaf age, plant developmental stage and the nature of the stress modulate the sulphur responses.

Proteases play a main role in the mobilization of storage proteins during seed germination. Until today, there is little information about the involvement of serine proteases, particularly subtilases, in the germination of barley grains. The aims of the present work were to study the contribution of serine proteases to the total proteolytic activity induced during germination of barley grains and evaluate the specific involvement of subtilases in this process. Proteolytic activity assayed against azocasein in the presence of specific inhibitors, showed that serine proteases contributed between 10 and 20% of total activity along germination. Subtilase activity increased from day 1 after imbibition with a peak between days 4-5. Moreover, in vivo determination of subtilase activity in germinating grains revealed increasing activity along germination mainly localized in the seed endosperm and developing rootlets. Finally, the expression of 19 barley genes encoding subtilases was measured by real time PCR during germination. Three of the analyzed genes increased their expression along germination, five showed a transient induction, one was down-regulated, nine remained unchanged and one was not expressed. The present work demonstrates the involvement of subtilases in germination of barley grains and describes the positive association of eight subtilase genes to this process.

Occurrence of genus Fusarium as endophytes in roots of healthy barley plants cultivated in fields under two levels of phosphorous (P) was examined. F. avenaceum, F. graminearum, F. konzum, F. oxysporum, F. sacchari, F. scirpi, F. solani were isolated from asymptomatic surface sterilized root tissues. F. konzum and F. solani were only present in the roots of fertilized plants, whereas F. sacchari was present in unfertilized plants. F. scirpi was isolated in highest frequency and relative density, and was higher under P deficiency suggesting that P availability would determinate the occurrence of this species, suggesting the possibility of their association with stressed plants. This is the first report on the occurrence of Fusarium spp. as endophytes in healthy barley plants. However, it remains to be determined whether Fusarium spp. isolated are nonpathogenic forms and beneficial to barley plants or latent pathogens that could produce disease symptoms in other conditions.

Subtilases are one of the largest groups of the serine protease family and are involved in many aspects of plant development including senescence. In wheat, previous reports demonstrate an active participation of two senescence-induced subtilases, denominated P1 and P2, in nitrogen remobilization during whole plant senescence. The aim of the present study was to examine the participation of subtilases in senescence-associated proteolysis of barley leaves while comparing different senescence types. With this purpose, subtilase enzymatic activity, immunodetection with a heterologous antiserum and gene expression of 11 subtilase sequences identified in barley databases by homology to P1 were analyzed in barley leaves undergoing dark-induced or natural senescence at the vegetative or reproductive growth phase. Results showed that subtilase specific activity as well as two inmunoreactive bands representing putative subtilases increased in barley leaves submitted to natural and dark-induced senescence. Gene expression analysis showed that two of the eleven subtilase genes analyzed, HvSBT3 and HvSBT6, were up-regulated in all the senescence conditions tested while HvSBT2 was expressed and up-regulated only during dark-induced senescence. On the other hand, HvSBT1, HvSBT4 and HvSBT7 were down-regulated during senescence and two other subtilase genes (HvSBT10 and HvSBT11) showed no significant changes. The remaining subtilase genes were not detected. Results demonstrate an active participation of subtilases in protein degradation during dark-induced and natural leaf senescence of barley plants both at the vegetative and reproductive stage, and, based on their expression profile, postulate HvSBT3 and HvSBT6 as key components of senescence-associated proteolysis.

The phloem transport of amino acids is a key step in the efficient use of nitrogen (N). Despite the importance of this issue, little information is known about the regulation of phloem transport of amino acids in plants with low phosphorus (P) supply and even less in relation to N availability. To this end, we studied not only the assimilate partitioning in young barley plants grown with low N or/and P supply, but also we analyzed the implications of the different isoforms of glutamine synthetase, cytokinin oxidase/dehydrogenase 2 and several senescence-related proteases. Our results demonstrated that low P supply causes an accumulation of different nitrogenous compounds in expanded leaves depending on N availability and an inhibition of the phloem exudation rate of amino acids only in high-N plants, indicating an interaction between N and P in the establishment of N-partitioning. The accumulation of nitrogenous compounds in leaves of low-P plants without the accompaniment in amino acid export to the phloem was not related to an increase in nitrate assimilation pathway neither with the modulation of glutamine synthetase 1_1 expression, as it had been observed for N availability. But, these results could be explained as a consequence of a delay in the transition from sink to source of leaves, thus keeping the older leaves as sink organs, as indicated by the increase in cytokinin oxidase/dehydrogenase 2 expression and the repression of several senescence-related proteases in low-P plants with good availability of N.

Understanding the way in which N and P availability affects the transport of sugar and amino acids is crucial to improve grain quality and yield. Thus, in the present study, two greenhouse and field experiments were conducted with barley plants grown with different N and P availabilities to assess the dynamics of the phloem transport of assimilates in relation to the beginning of flowering and senescence. The phloem transport of assimilates decreased before the beginning of protein degradation in all treatments, but the onset of flowering and senescence varied according to the N and P availability, as evidenced by the concentrations of proteins, amino acids, and sugar and the gene expression of senescence-related proteases and all glutamine synthetase isoforms. In N-deficient plants, the phloem transport decreased before flowering, but only when P was not limiting; in N- and P-sufficient plants it decreased at flowering; and in P-deficient plants it decreased after flowering. Therefore, only N-deficient but P-sufficient plants have a post-anthesis period with high export rate of assimilates. This alteration of phenology in relation to phloem leads to important consequences in assimilate utilization, as shown by the higher yield and N content of the former compared to P-deficient plants.

Sulphur (S) is incorporated into diverse primary and secondary metabolites that play important roles in proper growth and development of plants. In cereals, a fraction of the nitrogen (N) accumulated in developing grains is guaranteed by amino acid remobilization from vegetative tissues, a contribution that becomes critical when soil nutrients are deficient. Glutamine synthetase (GS) and amino acid transporters (AAT) are key components involved in N assimilation and recycling. The aim of the present study was to evaluate the effect of S availability on the expressions of HvGS and several selected HvAAT genes in barley plants and on the phloem exudation rate of amino acids. To this end, two independent experiments were designed to impose low S availability conditions to barley plants. Low S availability caused a decrease in the phloem exudation rate of amino acids as well as in the gene expression of all the HvGS genes and five of the six HvAAT genes analyzed. The strong correlation found between the phloem amino acid exudation rate and HvGS1-1, HvGS1-2, HvAAP7, and HvProT1 gene expression may indicate the participation of these genes in the regulation of amino acid remobilization through the phloem.

Fil: Veliz, Cintia Gabriela. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Oficina de Coordinacion Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agricolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomia. Instituto de Investigaciones en Biociencias Agricolas y Ambientales; Argentina

The commercial quality of malting barley is dependent on the content and composition of grain proteins which are subjected to nitrogen (N) and sulfur (S) control. In nutrient deficient soils, grain protein content is mainly a consequence of the remobilization efficiency. In order to evaluate the effect of N and S soil availability on phloem amino acid and sugar export rates and B- and C-hordein gene expression during grain filling, a factorial combination of N and S fertilization assay was carried out under field conditions. Besides, several carbon (C) and N metabolites were analyzed in leaves and spikes. We observed that, even under soil N and S availabilities that do not limit yield, N and S fertilization induced significant changes in N and C metabolism. N phloem remobilization was promoted by S fertilization independently of N fertilization and C remobilization was also promoted by S fertilization but only in N fertilized plants. The B- and C-hordein gene expression correlated positively with sugar and amino acid exudation rate, respectively. Our results suggest an important role of the export rate of sugars and amino acids in the regulation of grain prolamine expression.

An essential goal for modern agriculture is the simultaneous improvement of productivity efficiency and nutrient use efficiency. One way to achieve this goal in crops is to enhance nitrogen (N) and phosphorus (P) acquisition through the mycorrhizal association. This study examined the effect of mycorrhization on post-anthesis N and P dynamics and its impact on grain yield and quality in barley. In addition, the efficiency of both N and P utilization and remobilization was evaluated. With those purposes, barley plants inoculated or not with Rhizophagus intraradices were grown in a soil poor in N and P under greenhouse conditions. Inoculation with R. intraradices in barley enhanced both N and P content in grain and vegetative tissue and reduced phloem amino acid export rate. On the other hand, both N and P vegetative tissue content and phloem amino acid and P export rates decreased during grain filling, whereas N and P grain content increased in both treatments according to the senescence process. However, whereas N grain concentration decreased during grain filling, P grain concentration did not vary, thus suggesting a differential regulation on grain filling. Inoculation with R. intraradices improved the yield and grain quality, thus demonstrating that inoculation with R. intraradices in barley is beneficial, but mycorrhization caused a diminution in nutrient utilization efficiency. As the phloem remobilization rate of amino acids and P did not decrease during grain filling in R. intraradices-inoculated plants compared to non-inoculated ones, these results suggest that nutrient utilization efficiency is most probably regulated by sink strength rather by a mycorrhizal effect.

Senescence is the final developmental stage of every plant organ, which leads to cell death. It is a highly regulated process, involving differential gene expression and outstanding increment in the rate of protein degradation. Senescence-associated proteolysis enables the remobilization of nutrients, such as nitrogen (N), from senescent tissues to developing organs or seeds. In addition to the nutrient recycling function, senescence-associated proteases are also involved in the regulation of the senescence process. Nearly, all protease families have been associated with some aspects of plant senescence, and numerous reports addressing the new identification of senescence-associated proteases are published every year. Here, we provide an updated report with the most recent information published in the field, focusing on senescence-associated proteases presumably involved in N remobilization.

The induction of two subtilisin-like proteases (P1 and P2) associated with stress-induced senescence in young plants was investigated in adult wheat plants during the grain-filling period. Western blot analysis of flag leaf extracts showed that P1 was induced very late in the life cycle of the plants (9 days post-anthesis) and that 7 days later it reached a 2.5-fold increase with respect to the initial value at anthesis. On the other hand, the P2 signal was already detected previous to anthesis and increased soon after anthesis, reaching a fourfold increase by the end of the grain-filling period. The induction of P1 and P2 temporally correlates with the degradation of the Rubisco small and large subunits in the flag leaf, as well as with nitrogen (N) accumulation in the ears. At the same time, a decrease in the endogenous concentration of the cytokinins isopentenyladenine and isopentenyladenosine (iP + iPA) in the leaves was observed. In detached leaves senescing in the dark, the levels of both proteases were affected by 6-benzylaminopurine application: the induction of P1 was completely prevented, whereas the induction of P2 was reduced. Our findings demonstrate that both P1 and P2 are expressed in leaves of adult plants and are induced during natural senescence. These results enable us to postulate their participation in N remobilization to developing grains during monocarpic senescence and their regulation by a cytokinin-mediated mechanism.

The possible regulation of amino acid remobilization via the phloem in wheat (Triticum aestivum L.) by the primary enzyme in nitrogen (N) assimilation and re-assimilation, glutamine synthetase (GS, E.C. 6.3.1.2) was studied using two conditions known to alter N phloem transport, N deficiency and cytokinins. The plants were grown for 15 days in controlled conditions with optimum N supply and then N was depleted from and/or 6-benzylaminopurine was added to the nutrient solution. Both treatments generated an induction of GS1, monitored at the level of gene expression, protein accumulation and enzyme activity, and a decrease in the exudation of amino acids to the phloem, obtained with EDTA technique, which correlated negatively. GS inhibition by metionine sulfoximide (MSX) produced an increase of amino acids exudation and the inhibitor successfully reversed the effect of N deficiency and cytokinin addition over phloem exudation. Our results point to an important physiological role for GS1 in the modulation of amino acids export levels in wheat plants.

Nitrogen (N) remobilization in wheat (Triticum aestivum) plants is crucial because it determines the grain protein concentration and the baking quality of flour. In order to evaluate the influence of cytokinins on N remobilization during N starvation, we analyzed various N remobilization parameters in wheat plants that were watered with 6-benzylaminopurine (BAP) either with or without KNO3. Besides, the effects of BAP on protein synthesis were evaluated, and the size and ultrastructure of chloroplasts of BAP-treated plants were studied. BAP supply inhibited N remobilization of plants independently of N supply as shown by the increase in protein, Rubisco, chlorophyll, sugar and starch concentrations in the older leaves, the decrease in amino acid and sugar export to the phloem, and the decrease in protein, Rubisco and chlorophyll concentrations in the younger leaves. Besides, BAP supply increased nitrate reductase activity and decreased nitrate concentration, thus suggesting an increased assimilatory capacity. The increase in protein concentration could be explained mainly by a significant decrease in protein degradation and, to a lesser extent, by an increase in protein synthesis. Finally, an increase both in the size of the chloroplast and in the plastoglobuli and starch contents in BAP-supplied plants was observed. We propose that cytokinins retain the sink activity of the older leaves by inhibiting amino acid and sugar export to the phloem and stimulating assimilate accumulation in the chloroplasts of the older leaves. Besides, BAP may increase protein concentration of the older leaves both by decreasing protein degradation and maintaining protein synthesis even under stress conditions.

Solar ultraviolet-B radiation (UV-B) can have large impacts on the interactions between plants and herbivorous insects. Several studies have documented effects of UV-B-induced changes in plant tissue quality on the feeding performance of insect larvae. In contrast, the effects of UV-B-induced plant responses on the behavior of adult insects have received little attention. We carried out a series of field and glasshouse experiments using the model plant Arabidopsis thaliana L. and the crucifer-specialist insect Plutella xylostella L. (diamondback moth) to investigate the effects of UV-B on natural herbivory and plant–insect interactions. Natural herbivory under field conditions was less severe on plants exposed to ambient UV-B than on plants grown under filters that attenuated the UV-B component of solar radiation. This reduced herbivory could not be accounted for by effects of UV-B on larval feeding preference and performance, as P. xylostella caterpillars did not respond to changes in plant quality induced by UV-B. In contrast, at the adult stage, the insects presented clear behavioral responses: P. xylostella moths deposited significantly more eggs on plants grown under attenuated UV-B levels than on plants exposed to ambient UV-B. The deterring effect of UV-B exposure on insect oviposition was absent in jar1-1, a mutant with impaired jasmonic acid (JA) sensitivity, but it was conserved in mutants with altered ethylene signaling. The jar1-1 mutant also presented reduced levels of UV-absorbing phenolic compounds than the other genotypes that we tested. Our results suggest that variations in UV-B exposure under natural conditions can have significant effects on insect herbivory by altering plant traits that female adults use as sources of information during the process of host selection for oviposition. These effects of natural UV-B on plant quality appear to be mediated by activation of signaling circuits in which the defense-related hormone JA plays a functional role.

A senescence‐specific protease accounting for almost 70% of the total peptide hydrolytic activity of protein extracts, was isolated from detached wheat leaves induced to senescence by incubation in the dark for 72 h. Purification to apparent homogeneity was performed by ammonium sulphate precipitation, ion exchange chromatography and gel filtration chromatography. The enzymatic activity was followed by its ability to hydrolyse the synthetic peptide Suc‐AAPF‐pNA. SDS/PAGE and gel filtration analysis indicated that the enzyme was a dimer composed of two identical subunits of 59 kDa. The apparent Km and Vmax for the peptide were 1.18 mm and 2.27 mmol pNA mg−1 h−1, respectively. The enzyme was active at pH values above 8.0 and remained active after heat treatment at 60°C for 10 min. It was inhibited by chymostatin, indicating that the enzyme possesses a chymotrypsin‐like activity. Rubisco was readily hydrolysed by the purified protease. A sequenced internal fragment of 17 amino acids showed a high level of similarity (65–75% identity) with a highly conserved region of several plant subtilisin‐like serine proteases. The absence of this enzymatic activity in fractionated extracts from non‐senescent tissues suggests that it might play a role in the senescing process.

Grain protein content is one of the major determinants of the baking and nutritional quality of wheat. It has previously been reported that the ditelosomic line of wheat (Triticum aestivum L.) CSDT7BL, where the short arm of chromosome 7B is missing, shows a lower grain protein concentration than the normal line, but a similar grain yield. In the present paper the growth and nitrogen (N) metabolism of wheat plants cv. Chinese Spring (CS) and its ditelosomic line CSDT7BL were compared. When plants were grown to maturity in pots with different N supplements, the wild-type line showed a higher grain protein concentration and a lower straw N concentration than the ditelosomic line at every N level analysed, suggesting a deficiency in the N remobilization capacity. When 15-d-old plants were grown in a growth cabinet in pots with sand, and supplied with nutrient solutions of different nitrate concentrations, the ditelosomic line showed no differences in N uptake per unit of root dry weight, nitrate reductase activity, nitrate, total N concentration or free amino acid concentration. However, the ditelosomic line showed a decreased capacity to export amino acids in the phloem under high N, independently of the N source. This deficiency was also observed under dark-induced senescence. The diminished export of amino acids to the phloem was principally caused by a decrease in the export of Glu, Asp, and Gln. It is suggested that the decrease in grain protein concentration in the ditelosomic line is a consequence of defective export in the phloem of these amino acids.