Metrics details The systemic coordination of accumulation of plasma membrane aquaporins (PIP) was investigated in this study in relation to mycorrhized maize response to a rapid development of severe drought followed by rewatering followed by a transient increase under rewatering mycorrhiza contributed to maintenance of high and stable levels of PIPs in both plant organs after an initial increase Isoelectric focusing electrophoresis resolved up to 13 aquaporin complexes with highly reproducible pl positions across leaf and root samples Mass spectrometry recognized in leaves and roots a different ratio of PIP1 and PIP2 subunits within 2D spots that accumulated the most drought regulation of aquaporins in roots was manifested as the prevalence of complexes that comprise almost exclusively PIP2 monomers the leaf response involved enrichment in PIP1s PIP1s are thought to enhance water transport facilitate CO2 diffusion but also affect stomatal movements might explain a stress tolerance mechanism observed in mycorrhizal plants resulting in faster recovery of stomatal water conductance and CO2 assimilation rate after drought the stoichiometry within heterocomplexes consisting of proteins of each group may differ significantly in tissues from distant organs studies are needed to reveal aquaporin interactions in plant cells and to determine the physiological relevance of these processes also in the context of mycorrhizal cooperation mycorrhizal (AM) plants showed a much faster reversal of drought-induced leaf senescence and CO2 fixation rate than their non-mycorrhizal (NM) counterparts In the present study we test the hypothesis that rapid recovery of AM maize from deep water deficits is linked to symbiotic regulation of maize aquaporins we argue that the ratio of PIP1 and PIP2 monomers that form aquaporin heterocomplexes corresponds to the patterns of their tissue-specific accumulation under given hydration conditions and their abundance can be altered by the presence of mycorrhiza All research under this study was conducted according to Polish national law and did not require any additional permits We designed a rapid development of water stress to minimize leaf senescence progression related to drought-altered nutrients availability Well-watered plants were removed from pots and transferred to the cabinet with low air humidity (30%) The mixture of coconut fiber and sand in which the roots remained immersed helped to obtain severe but reversible drought effects in as little as 7 days Soil drought was imposed by stopping irrigation for 7 days to achieve a severe drop in plant water potential ( leaves < -1.5 MPa) followed by renewed fertilizer irrigation for 5 days until complete rehydration of plant tissues the level of mycorrhizal colonization was achieved in the present study at the similar level as previously arbuscular abundance in colonized parts of root fragments at the time of experiments (12 weeks after sowing The evaluation of leaf physiology was carried out in middle leaves (the ear leaf and the leaf above the cob) SDS-PAGE electrophoresis was performed on 11% polyacrylamide gels according to the TGX Stain-Free method (BioRad) 10 µg of protein samples were mixed in a 1:1,6 ratio (v/v) with solubilization buffer (96 mM Tris-HCl pH 6.8 The semidry blotter system (Merck) was then used to transfer proteins to Immobilon-P membrane Immunochemical identification was carried out with anti-PIP1;1–3 (Agrisera AS09 489) and anti-PIP2;1–7 (Agrisera The membrane was blocked overnight at 4 °C in 3% BSA in PBS and then incubated for 1 h in RT with PIP1;1–3 antibody at 1:1000 dilution and with PIP2;1–7 antibody at 1:3000 dilution in PBS-T buffer containing 1% BSA The membrane was then incubated for 1 h in RT with the secondary antibodies (Agrisera Antigens were detected with Lumi-Light Western Blotting Kit (Roche) For estimation of PIP monomers composition according to coordinates determined by immunodetection on parallel gels and then subjected to tandem mass spectrometry procedure The microsomes were treated with Brij-58 detergent to delipidate and remove surface-associated proteins41 240 µl 2% Brij-58 and 4 µl PIC were gently mixed for 30 min at 4 °C After overnight precipitation at -26 °C with 10% (w/v) TCA in acetone and 0.07% (v/v) β-mercaptoethanol the proteins were pelleted for 15 min at 20,000 g and then washed three times with pure acetone and 0,07% β-mercaptohetanol acetone was removed and the precipitate was dried for 10 min The solubilization of integral membrane proteins was carried out by suspending 120 µg of protein in 120 µl of IEF buffer containing: 7 M urea IPG strips were placed for 15 min in 10 ml of pH equilibrating buffer: 65 mM DTT 50 mM Tris-HCl pH 6,8 and then alkylated for 15 min in a buffer containing 2,5% iodoacetamide instead of 65 mM DTT For protein separation according to their molecular masses the strips were subjected to Stain-Free SDS-PAGE as above 250 µg of microsomes were solubilized by adding 2% ASB-14 and 0.1% TX-100 detergents suspended in 300 µl of TBS in the presence of 1.7% PIC The sample was incubated for 2 h at 4 °C with stirring and then centrifuged for 10 min at 110,000 g The supernatant was collected and diluted 1:1 with TBS to obtain the final 1% concentration of ASB detergent The samples were agitated overnight at 4 °C with 1 µL of PIP2;1–7 antibodies Antigen-antibody complexes were bound to Protein A-Sepharose 4B bed (Merck) washed and then eluted by incubation with 1.6x sample buffer (20mM Tris pH 6.8 The supernatant after the following centrifugation was quenched with 2 ml of cold acetone with 0.07% β-mercaptohetanol and precipitated as described above The obtained pellet was dried in a fume hood for 30 min and then dissolved in 100 µl buffer: 4 M urea The final acetone concentration was 7 M to avoid the risk of precipitation of urea present in the sample buffer The experimental data are presented as means of a specified number of replicates (n) ± SEM Statistical analysis was carried out using the Student’s t-test for groups with normal distribution (Shapiro-Wilk test) one-way or two-way analysis of variance (ANOVA) and Tukey-Kramer multiple comparison test Statistically significant results were those for which the level of statistical significance value (p) reached: p ≤ 0,05 (*) Effect of fast drought development (S0 R5) on plant PSII quantum conversion efficiency [(A) Rfd and (C) Fv/Fm] and (D) leaf nitrogen balance index (NBI) of mycorrhizal (AM) and non-mycorrhizal (NM) maize The bars represent average values taken from the middle leaves (ear leaf and the leaf above) in the upper half of leaf length The error bars show the standard error of the mean (NBI: n = 100 5 plants x 2 leaves x 10 measurement points; RFd and Fv/Fm: n = 14 No statistically significant differences between mean values for NM and AM plants were found Lowercase or uppercase letters indicate significant differences (p < 0.05) among stress time points for plants of the same symbiotic status (one-way ANOVA) Measurements of leaf gas exchange parameters exposed a compensatory effect of mycorrhiza because a faster restoration of photosynthetic efficiency occurred in the course of rewatering (Table 1) and transpiration (Tmax) strongly decreased these activities were fully restored in AM plants while NM plants did not reach more than 50% of the initial efficiency These results indicate that high fertilization before stress and the imposing of fast soil drought could eliminate fungal adjustments in leaf physiology during the progression of the plant water deficit but left a variation in the Amax and gs parameters at the time of rewatering Light-saturated CO2 assimilation rate (Amax µmol m− 1) of mycorrhizal (AM) and non-mycorrhizal (NM) plants measured in the main phases of the water treatment: optimal watering (S0) Asterisks indicate significant differences (p < 0.05) between AM and NM plants on particular days of water treatment (Student’s t-test The molecular marker was cut-of prior to incubation of membranes with antibodies and stained using Coomassie Blue For further information see Supplementary data The establishment of the above-described cultivation regime allowed us to proceed with analyses aimed at demonstrating whether the overall level of aquaporin accumulation in root and leaf cells is subject to change in correlation with the symbiotic status and rapidly changing irrigation conditions Of particular interest was the rehydration period during which mycorrhiza was found to have a compensatory effect on transpiration and photosynthetic efficiency Aquaporin immunodetection under drought treatment (Fig. 2, top marked in red and recovery patterns underlined in blue) was further quantified by densitometry and averaged (Fig. 3) opposite patterns of root and leaf aquaporin accumulation were observed in response to water treatment both PIP1s and PIP2s were highly abundant under full hydration (S0) while during drought (S7) their level dropped to about 50% and 25% whereas protein levels increased 4–5-fold as severe drought developed Lowercase or uppercase letters indicate significant differences (p < 0.05) among stress time points for plants of the same symbiotic status Asterisks indicate significant differences (p < 0.05) between AM and NM plants on particular days of water treatment (two-way ANOVA with Tukey’s post-hoc test Aquaporins are membrane-spanning proteins and therefore are more challenging to analyse by proteomic methods than soluble proteins because of their hydrophobic properties we have managed to develop a solubilization procedure that allows effective separation of functional PIP dimers from the microsomal fraction This indicated the presence of PIP complexes in both roots and leaves with very similar enrichment in post-translational modifications Samples taken from fully irrigated variants (S0) and from day 3 of rehydration (R3) were selected for this purpose Drought (S7) or initial irrigation period (S0) was accompanied by a strong down-regulation of the level of PIP proteins in the roots or leaves these variants were omitted from the analysis presented In root samples taken during full hydration (S0) the regulation of aquaporins did not appear to favour any of the IEF isoforms although the level of accumulation of individual complexes was much higher in non-mycorrhizal plants we did not analyse samples from the drought period (S7) we observed selective persistence of oligomers in the range of pI 7.2–7.7 parallel to the almost complete removal of some complexes in the range of pI 8.5–9.2 regardless of the symbiotic status and irrigation level the highest abundance was attributed to the IEF complexes located at the pI positions 8.1 to 9.2 When analysing independent SDS-IEF replicates (not shown) it was not possible to identify IEF complexes whose accumulation would be prominent in response to drought or irrigation restoration or symbiotic variants The MS identification showed that the ratio of PIP1 and PIP2 monomers was more or less equal in spots located at the pI positions 8.1 to 9.2 the proportion of PIP1s remained significantly lower it appears to be partitioned to a greater extent by cell-to-cell water flow The risks and factors described above could also affect AM plants but, as summarized in Table 2 the symbiotic regulation of PIPs followed a drought temporal pattern clearly different from that recorded in NM plants: (i) AM root samples taken under fully hydrated conditions contained about 25–35% less aquaporins than NM roots; (ii) drought had a much lower impact on PIP accumulation in AM roots compared to a significant drop in NM roots; (iii) AM roots showed an incremental accumulation of PIPs against the whole sequence of water availability in contrast to the sinusoidal pattern found in NM roots; and (iv) after rewatering AM leaves retained an elevated amount of PIP proteins longer than NM leaves Considering features (ii) and (iii) of AM plants their roots appeared to be capable of engaging a positive feedback mechanism while the response of NM plants was based on a negative feedback scheme a similar positive regulation of PIPs distinguishes the leaf response of AM plants Although PIPs also accumulated in NM leaves during the drought phase mycorrhiza maintained this effect when irrigation was restored apparently contributing to a faster recovery of leaf gas exchange both at the transcriptional and post-translational levels when a plant is exposed to a certain stress or change in the environment no attempt has been made to determine the effect of drought on the variation in monomers’ association within aquaporin oligomers and their accumulation when a mycorrhizal partner is involved in water conduction mechanisms This feature allowed us to design the procedure to reveal the presence of functional aquaporin dimers under the assumption that the stability of disulphide bridges is preserved during the isoelectric focusing step of two-way electrophoresis We assumed that we could identify these dimers if they differed in pI values due to the different associations of the PIP1 and/or PIP2 monomers and the associated post-translational modification who showed that phosphorylation of aquaporins rather than an increase in their synthesis is a mechanism correlated with the increase in root cell water transport capacity in the presence of mycorrhiza we did not achieve sufficient efficiency in identifying phosphorylated residues to formulate conclusions about the post-translational modifications of individual 2D spots in response to the imposed water conditions (see comments on possible effect of phosphorylation on IEF spot distribution in the supplementary data file) It refers to the fact that root-abundant oligomers separated in the pI range 7.2–7.7 showed a very low abundance of PIP1-type isoforms Such a disproportion between PIP1 and PIP2 isoforms was not present in leaf-abundant complexes located at pI positions 8.1 to 9.2 The proposed systemic coordination scheme for the regulation of PIP aquaporin in response to re-watering after drought leaf gas exchange and overall PIPs abundance are shown according to the results obtained we illustrate enrichment in PIP1 or PIP2 monomers specific to the most abundant aquaporin heteromers present in leaf or root samples The tissue-specific composition was approximated based on the emPAI ratio of PIP1;2 to PIP2;1 isoform estimated by MS/MS Mycorrhizal regulation during rehydration manifested in roots primarily as accumulation of PIP2-rich oligomers Such a composition can result in lower water transmembrane conductivity than that involving PIP1 as predicted by experimental and theoretical models This may allow a more effective counterbalance against the risk of root water loss Considering enrichment of leaf aquaporins in PIP1s subunits and their contribution to water and CO2 diffusion and controlling stomatal 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Plant. Cell. Physiol.45, 823–830. https://doi.org/10.1093/pcp/pch120 (2004) Interactions between plasma membrane aquaporins modulate their water channel activity FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization Plant aquaporins: from transport to signaling Alexandersson, E. et al. Transcriptional regulation of aquaporins in accessions of Arabidopsis in response to drought stress. Plant. Journal: Cell. Mol. Biology. 61, 650–660. https://doi.org/10.1111/j.1365-313X.2009.04087.x (2010) Boursiac, Y. et al. Early effects of salinity on water transport in Arabidopsis roots. Molecular and cellular features of aquaporin expression. Plant. Physiol.139, 790–805. https://doi.org/10.1104/pp.105.065029 (2005) Fouquet, R., Leon, C., Ollat, N. & Barrieu, F. Identification of grapevine aquaporins and expression analysis in developing berries. 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P., Scoffoni, C. & Sack, L. How does Leaf Anatomy Influence Water Transport outside the Xylem? Plant. Physiol.168, 1616–1635. https://doi.org/10.1104/pp.15.00731 (2015) In the Leaf: A Platform for Performing Photosynthesis81–96 (Springer PIP water transport and its pH dependence are regulated by tetramer stoichiometry Heteromerization of PIP aquaporins affects their intrinsic permeability Are aquaporins expressed in Stomatal complexes Promising targets to Enhance Stomatal dynamics Yaneff, A., Vitali, V. & Amodeo, G. PIP1 aquaporins: intrinsic water channels or PIP2 aquaporin modulators? FEBS Lett.589, 3508–3515. https://doi.org/10.1016/j.febslet.2015.10.018 (2015) Yepes-Molina, L., Barzana, G. & Carvajal, M. Controversial regulation of Gene expression and Protein Transduction of Aquaporins under Drought and salinity stress. Plants (Basel). 9https://doi.org/10.3390/plants9121662 (2020) A proteomic study reveals novel insights into the diversity of aquaporin forms expressed in the plasma membrane of plant roots Single mutations in the transmembrane domains of maize plasma membrane aquaporins affect the activity of monomers within a heterotetramer Mesophyll diffusion conductance to CO2: an unappreciated central player in photosynthesis In the leaf: A Platform for Performing Photosynthesis 163–208 (Springer Arbuscular mycorrhizal symbiosis increases relative apoplastic water flow in roots of the host plant under both well-watered and drought stress conditions Scoffoni, C., Sack, L. & Ort, D. The causes and consequences of leaf hydraulic decline with dehydration. J. Exp. Bot.68, 4479–4496. https://doi.org/10.1093/jxb/erx252 (2017) Download references We would like to thank Maksymilian Chmielewski for help with depositing mass spectrometry proteomics data in the PRIDE database The publication was co-financed within the National Science Centre Poland (Project Number 2011/01/B/NZ9/00362 to WP) and the European Union under the European Social Fund (Project Number DFS.VI.052.4.17.6 to EP-L) Ewelina Paluch-Lubawa & Władysław Polcyn designed and performed the experiments; E.P.-L analyzed the data and created the figures and tables; E.P.-L The authors declare no competing interests Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Below is the link to the electronic supplementary material Download citation DOI: https://doi.org/10.1038/s41598-024-72828-8 Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Sign 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Volume 12 - 2021 | https://doi.org/10.3389/fpls.2021.635619 This article is part of the Research TopicPlant Responses to the Dark ScenarioView all 11 articles This review synthesizes knowledge on dark-induced barley leaf senescence (DILS) as a model and discusses the possibility of using this crop system for studying senescence and autophagy mechanisms It addresses the recent progress made in our understanding of DILS The following aspects are discussed: the importance of chloroplasts as early targets of DILS the role of Rubisco as the largest repository of recoverable nitrogen in leaves senescing in darkness morphological changes of these leaves other than those described for chloroplasts and metabolic modifications associated with them DILS versus developmental leaf senescence transcriptomic differences and finally the observation that in DILS autophagy participates in the circulation of cell components and acts as a quality control mechanism during senescence the symptoms of degradation can be reversed the question also arises how plant cells regulate stress-induced senescence via autophagy and how the function of autophagy switches between cell survival and cell death DILS program setup are barley seedlings grown in growth chamber for 7 days under controlled conditions (day/night 16/8 h light intensity 150 μmol m–2 s–1 Pots with seedlings on seventh day of growth are transferred to dark conditions to initiate senescence Table 1. Overview of the experimental setups of dark-induced barley leaf senescence assays employed through the different studies cited in this review, and comparison of assessed parameters against DILS (Sobieszczuk-Nowicka et al., 2018) This review will address the studies that allow showing (i) the survival strategy behind dark-induced senescence in barley plant and (ii) dark-induced barley leaf senescence to be used as a model referred to in the manuscript also as DILS program The idea of a program as applied to living systems has been taken from computer science always starts and fails in more or less the same manner and physiological data that reveal events in barley DILS program the time limit for dark-to-light transition for reversal of the senescence process and progression of senescence through autophagy into the PCD phase a 2-day period of light re-exposure does not suffice to return the level to that of the light control in spite of the high potential of chloroplasts to restore the photochemical efficacy of solar energy conversion the energy conversion of excitation and/or the use of potential energy may be restricted by some unidentified factor whose reversibility is compromised which also is overexpressed in later stage of DILS The efficiency of regulation of autophagic processes is a symptom of the vitality of senescing cells which at each stage must hold the ability to maintain homeostasis we suggest that a critical step that determines the point of no return in DILS model is macroautophagy control This suggests the participation of NAC transcription factors as regulators of a range of processes in plant development and stress responses There has been developed a crop model that demonstrates and explains early and late events of DILS and identifies the time limit for dark to light transition for reversal of the induced-senescence process within a leaf – DILS The first phase is more strongly emphasized by cessation of photosynthesis Disintegration of chloroplasts correlated with the degradation of Rubisco and PsbA-D1 proteins Despite the advanced state of macroautophagy in this phase the processes of degradation turned out to be reversible The reversal of DILS program involves regaining photosynthesis and increase of chlorophyll content and it takes place irrespectively of the activation of ATG genes is characterized by irreversibility of senescence and its progression into PCD exemplified by the involvement of both autophagy and PCD pathways chromatin condensation accompanied with nDNA fragmentation Non-invasive methods for quantifying photosynthetic efficiency and barley leaf nitrogen status established the time frame during which DILS enters the irreversible phase Rfd is determined there as the earliest parameter that correlated well with the cessation of photosynthesis together with the appearance of micro-autophagy symptoms DILS program is also found to be characterized by the upregulation of processes that enable the recycling of degraded metabolites in darkness and partial upregulation of glyoxylate and tricarboxylate acid cycles ES-N conceived the topic of the manuscript ES was responsible for the layout of the manuscript and prepared the table ES-N coordinated writing of the manuscript All authors listed have made a substantial direct and intellectual contribution to the work Poland (Project Numbers 2018/29/B/NZ9/00734 and 2018/30/E/NZ9/00827 to ES-N and 2017/27/N/NZ9/02135 to EP-L) The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest We thank Richard Ashcroft (bioscience editor) for the professional language editing of the manuscript. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Ewa Sobieszczuk-Nowicka, ZXZhYW5uYUBhbXUuZWR1LnBs Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish that their Lubawa USA subsidiary entered the final phase of proceedings for procurement of mobile that the order for multi-spectral camouflage was placed by the US Army part of the US Army proceedings for camouflage systems was placed via the HDT Global – Lubawa partner in the USA Multi-spectral, mobile camouflage manufactured by Miranda can be utilized on armoured vehicles, such as Rosomak APC. /Photo: Lubawa The purchase order is worth USD 468 000 (EUR 386 000) and was acknowledged in the late night hours Polish time, on the 26th of April. It`s the first contract of the CCP (Competitive Prototyping Phase) stage of the US Army tender for multi-year deliveries of camouflage systems. There are only 3 offerors present in this stage of proceedings. In the CCP stage, Lubawa will deliver the camouflage systems produced by Miranda subsidiary. The proceedings are to finish in 2018 with a choice of single provider. If HDT Global/Lubawa joint venture wins the tender, the production phase will start. The consortium would have to provide USD 480 mln (EUR 397 mln) worth of multi-spectral camouflage systems over the 10-year period. This phase would start in 2019 with the production being performed by HDT Global in the USA, under the Lubawa USA licence. This mobile camouflage is already in use by Finland`s Army and is utilized on 106 BMP-2MD personnel carries. The contract with Fins was signed in June 2016 and was worth PLN 4,5 million (EUR 1,06 million) Polish army uses the camouflage nets Berberys and Berberys-S, produced by Miranda. These are intended for stationary objects and for mobile objects when stationary. At the same time, the Armament Inspectorate is analysing the possibilities of purchase of the multi-spectral camouflage. The decision should be reached in 2018. Volume 10 - 2019 | https://doi.org/10.3389/fpls.2019.00496 This article is part of the Research TopicDrought and Salinity Tolerance in Mycorrhizal Plants View all 8 articles Under fertilization levels specific to intensive farming the impact of compensation of soil nutritional value by arbuscular mycorrhiza (AM) might be limited the question arises whether modern crop varieties are able to gain symbiotic benefits under other challenging field conditions in this study we aimed to evaluate the contribution of Rhizophagus irregularis to the drought response of a stay-green corn hybrid in pot cultures equally fertilized until silking compared to non-mycorrhizal (NM) counterparts The highest tested fertilization regime not detrimental to the long-term vitality of intraradical hyphae reached the levels recommended for field cultivation of silage corn except phosphorus application restricted to 60% mycorrhiza increased leaf nitrogen and phosphorus acquisition but only in cultures supplied with low NPK levels only the older leaves retained AM dependency whereas for other leaf positions the AM-NM differences were leveled out The similar size and nutritional status of highly fertilized AM and NM cultures eliminated fungal benefits before and during the 2-week drought progression mycorrhizal contribution became evident at the time of renewed watering when AM plants showed much faster reversal of drought-induced leaf senescence symptoms: impaired photosynthesis and nitrogen management Our results suggest that mycorrhiza can alter drought-induced senescence even in stay-green mutants this effect was apparently not mediated by AM-improved growth but triggered by activation of fungal transport at the time of recovery the fungal protective potential was shown to be preserved at the expense of lowering AM vesicle number It can be interpreted as engagement of hyphal nutritional resources targeted to maintain the symbiotic relationship despite the reduced vitality of the host we compared the productivity of AM and NM cultures subjected to short-term drought at silking time and further fertilized with moderate or high NPK doses until the grain-filling stage The yield and nutritive value of green forage showed that alleviation of drought-induced senescence by AM was not sufficient to have a significant positive effect on the final productivity compared to NM plants such a behavior is in common to AM plants of other species typically exhibiting greater photosynthetic rates under drought conditions in this study we aimed to evaluate the contribution of Rhizophagus irregularis to the drought response of a stay-green corn hybrid in pot cultures fertilized until silking with high NPK doses but also intending to limit differences in nutritional status of AM and NM counterparts To facilitate access to nutrients and prevent from uncontrolled fertilizer concentration changes we designed a soil-free semi-hydroponic system with a mixture of coconut fiber and sand and frequent irrigation with a water-soluble fertilizer The substrate mix has excellent water and air exchange properties allowing to obtain severe but quickly reversible soil drought effects we sought to resolve the following issues: (1) to define fertilization limits and optima for long-term intraradical hyphal vitality and symbiotic coexistence with corn (silage stay-green hybrid ‘Opoka’) until the early generative stage; adjusted to the nutritional status of NM counterparts would show alleviated leaf senescence after exposure to progressively increasing drought; (3) to evaluate if highly fertilized mycorrhizal cultures would present altered fodder quality after drought applied at the particularly sensitive time of silking Sterile germinated seeds of Zea mays (hybrid Opoka Poland) were planted in substrate trays with 25 ml of commercial sterilized peat substrate for vegetables Two week-old seedlings were transferred into 4 L pots (one plant per pot) filled with coconut fiber (Ceres International)/0.8–1.2 mm sand mixture (3:1 The experiments were carried out in phytotron under 16 h/8 h light and dark regime and respective temperatures of 25 and 21°C photosynthetic photon flux density of 900 μE m-2s-1 Pots were supplemented with tap water to compensate for daily cumulative evapotranspiration (ca 3 × 200 ml per week at the 8-leaf stage) In this water volume a fully soluble commercial fertilizer of lowered phosphorus content was applied (Kristalon Blue label 3 S and addition of microelements (%): 0.025 B; 0.07 Fe (DTPA); 0.04 Mn (EDTA); 0.025 Zn (EDTA); 0.004 Mo; 0.01 Cu (EDTA) The maximal fertilization level (denoted as 1xD) defined as not detrimental for long-term hyphae vitality was 114 mg N/36 mg P2O5/120 mg K2O/18 mg MgO as expressed in per plant weekly doses For doses of 0.5xD and 1xD the plants reached 180 cm height and 240 g weight 8 fully green leaves and flowering phase in the typical time for the variety The maximal fertilizer volume was reduced to 25% for the first 6 weeks (until the 6-leaf stage) in order to establish fungal colonization of roots After this period the plants were provided for 4 weeks (until tasseling 10 weeks after seeding) with one of four dilutions of 1xD dose (task 1) The visible symptoms of leaf N or P deficiency was specific mainly to 0.125xD fertilization variant and first 4 weeks cultivation on 0.25xD fertilizer dilution For drought experiments (task 2) the cultivation from 6th to 12th week was continued on 1xD fertilization level until silking (63 BBCH stage when pollination begins and ear silks begin to emerge) Soil drought was imposed by withholding watering for several days the pots were treated from 6th week with half (0.5xD) or maximal (1xD) fertilization volume until half-milkline stage of grain development recommended for silage harvest (85 BBCH stage symbiotic or drought treatments 4–6 plants The number of treatments and replicates was provided in legends of appropriate figures Each seedling was inoculated at the time of transfer with suspension of 250 spores of Rhizophagus irregularis. The inoculum (Centre for Mycorrhizal Research, The Energy and Resources Institute, New Delhi, India), free of microbiological contaminants, was obtained from monoxenic root organ cultures (Adholeya et al., 2005) For fodder quality analyses the plants were cultivated on the soil taken from preceding AM or NM cultures (i.e. keeping the same microbiological composition diluted 1:15 (w/w) with sterile coconut-sand substrate Vesicles abundance was quantified as an average number per cm of total root length Leaf samples were taken from three vertical canopy positions (counted from top): upper leaves (2nd and 3rd) middle leaves (ear leaf and leaf above the ear) and lower leaves (6th and 7th) For each of these positions the tissue material was collected avoiding the midrib The data represent averaged values (weight N or P units per leaf dry mass from two adjacent leaves (joined leaf numbers: L2+3 using four plants for each fertilization and symbiotic variant 0.5 – 1 g of leaves or roots was dried for 4 h at 103°C to a constant weight in accordance with the applicable AOAC standards A portion of plant material was used to determine total N concentration according to Kjeldahl method using Kjeltec 8400 Auto Sampler System The phosphorus content was analyzed after additional 10 h mineralization at 500°C according to ammonium molybdate colorimetric method (PN-ISO 6491) in conjugation with UV-visible Nicolet Evolution 300 spectrophotometer Leaf nitrogen status was estimated with chlorophyll/flavonoids ratio (NBI, Nitrogen Balance Index) using Dualex 4 Scientific (Force-A, Orsay, France) fluorimeter. The chlorophyll level was estimated from red light (710 nm) transmittance, automatically corrected for the interference from other leaf structures by division by transmittance at the reference wavelength of 850 nm (Cerovic et al., 2012) The amount of flavonoids is estimated from difference in chlorophyll fluorescence induced by UV (375 nm) and red light (650 nm) since only UV is affected by the presence of flavonoids The readings covered upper surface of apical half of two leaves from middle nodes (ear leaf and leaf above) and data was presented as the averaged values of 50–80 sampling points from four plants for each symbiotic and drought or fertilization variant The data was presented as the averaged values of 8 readings taken from the base part of two leaves from middle nodes (ear leaf and leaf above) from 4 plants for each symbiotic and drought or fertilization variant Measurements of AM and NM plants were made in a staggered manner during full irradiance period of phytotron light regime Maximum leaf gas exchange capacity (light-saturated photosynthetic rate μmol CO2 m-2 s-1 and maximum stomatal conductance to H2O mol H2O m-2 s-1) was determined with Q-Box CO650 Plant CO2 Analysis Package (Qubit Systems The stomatal aperture reached maximum after 20 min exposition to 3000 μmol m-2 s-1 photosynthetic photon-flux density readings were taken from the same 4 plants for each symbiotic and drought or fertilization variant and covered the base area of two middle leaves (9 cm2 of each sampling area) Measurements of mycorrhizal and NM plants were made in a staggered manner during full irradiance period of phytotron light regime Photosynthetic nitrogen (PNUE) or phosphorus (PPUE) use efficiency is the ratio of light-saturated CO2 assimilation rate to leaf N or P-content (expressed as weight units per leaf dry mass Midday leaf and root water potential was measured according to modified Porcel and Ruiz-Lozano (2004) method with a C52 thermocouple psychrometer (Wescor Three leaf disks about 5 mm in diameter taken from lower leaves and three 5 mm long fragments of secondary feeders roots were cut and stored as time samples in liquid nitrogen After thawing for 5 min at 35°C samples were sealed in the C-52 chamber necessary for vapor pressure stabilization within the chamber the readings were recorded by the Wescor PSYPROTM microvoltometer Relative water content (RWC) – normalized to DW ratio of fresh weight (FW) to weight of tissue in full turgor (TFW after 4 h of saturation at 4°C): RWC = [(FW-DW)/TFW-DW)] × 100 Chemical analysis of representative samples of fresh maize (chopped, mixed and dried stalks, leaves and ears) were done at the time of harvest, in accordance with the applicable AOAC2 standards or the Polish Standard in Department of Animal Nutrition Laboratory (Poznań University of Life Sciences from four separate treated plants for each fertilization and symbiotic variant Dry matter was analyzed in binder dryer according to AOAC 934.01 method. Crude protein (CP) and nitrogen content was measured by Kjeldahl method (AOAC 976.06) in Kjelfoss Automatic 16210. Crude fiber3 (CF) was analyzed by Tecator Foss Fibertec System M Ether extract (EE) was analyzed according to Soxhlet method (AOAC 2003.06) and Tecator Soxtec System HT 1043 Crude ash (Ash) was collected after a sample was burnt in Nobertherm oven (550°C) (AOAC 942.05) Nitrogen free extract was calculated on the basis of chemical composition according to equation NFE = (100 – CP – CF – EE – Ash) the nutritive value of maize silage was calculated on laboratory analysis of fresh maize using the PrevAlim 3.23 software (Educagri/INRA Analyses were conducted using the appropriate procedures (PROC MEANS Statistical analyses were performed using the general linear models procedure and Duncan’s multiple range test Differences were reported as significant when P ≤ 0.05 Effect of mycorrizae and fertilization level0 on nitrogen (N) and phosphorus (P) accumulation in leaves at three vertical canopy positions P and N content of non-mycorrhized (NM) and mycorrhized (AM) plants grown at four fertilization levels (0.125xD The data represent averaged values (n = 4) taken as mixed samples from two adjacent leaves (joined leaf numbers: L2+3 representing four plants for each fertilization and symbiotic variant The measurements were made at the time of tasseling (10 weeks after sowing) after 4 weeks fertilization with one of four fertilizer dilution The minerals content was expressed as weight units per leaf dry mass (g 100g-1) Asterisks represent statistical significance between NM and NM means according to t-Student test at p < 0.05 In roots, there was no tendency for P and N accumulation caused by fertilizer increase. The variances due to symbiosis were also small, as the average P content (% DW) was 0.24 ± 0.01 for NM and 0.27 ± 0.01 for AM, while the average N content was 1.09 ± 0.06 for both NM and AM. Leaf N and P absorption was studied at three leaf positions: upper, middle and lower (Figure 1 and L6 + 7 indicate pairwise collected leaves Leaf P and N content responded to fertilizer increase at each leaf position but the point of saturation was reached at 0.5xD fertilization level above which accumulation growth decelerated only the top two nutrition variants did not show visual N and/or P deficiency symptoms The lowest fertilization regime (0.125xD) was the only one with AM-dependent P accumulation significantly higher at each leaf position (Figure 1) The difference in P responsivity between AM and NM decreased with increasing dose under high fertilization only for the older leaves (L6+7) the mycorrhizal enhancement was observed whereas for upper or middle leaves the difference between AM and NM plants was not statistically significant Similar AM compensation for older leaves was related to N content Mycorrhiza increased leaf N acquisition mainly at lower fertilization levels but this effect became significant starting from the 0.25xD dose It means that the threshold for N availability was at least several-fold higher than for P The Rhizophagus irregularis vaccine was a suspension of fungal spores taken from monoxenic root cultures (TERI, New Delhi). The inoculum production involved optimization of spore germination under aseptic conditions, so it provided high intraradical hyphal growth and stable vitality. Figure 3A summarizes the parameters of fungal root colonization examined against a wide range of fertilizer dilutions applied until 10th week of corn culture growth The 1xD dilution (13.6 mM N and 0.86 mM Pi) was the highest fertilizer concentration found as not detrimental to long-term hyphal vitality and was eventually chosen for further drought experiments during the first 6 weeks after sowing (until the 6-leaf stage) the fertilizer volume had been reduced to 0.25xD level in order to prevent inhibition of fungal root infection Long-term intraradical hyphae vitality under different fertilization and drought regimes (A) Fertilizer or drought effect on hyphal root colonization and arbuscules abundance F% – frequency of fungal structures in the root system M% – intensity of the mycorrhizal colonization a% – arbuscular abundance in colonized parts of root fragments after 6 weeks on 0.25xD fertilizer dilution and additional 4 weeks on fertilization levels annotated in the table (10th week after sowing) Drought impact was examined after additional 2 weeks of drought and 1 week-long recovery; (B) Developmental stages and hyphae vitality 2 and 3: acceleration of arbuscules development between 6th and 8th week of growth on 0.25xD fertilization level 4: vesicles abundance after additional 4 weeks on 1xD dose 5: sporulation level at the time of silage harvest (20th week after sowing); (C) Effect of fertilizer dose on AM vesicles abundance after 6 weeks on 0.25xD fertilizer dilution and additional 4 weeks on fertilization levels annotated in the table (10th week after sowing); (D) Effect of drought and recovery on AM vesicles abundance after 6 weeks on 0.25xD fertilizer dilution and additional 4 weeks on 1xD fertilization levels annotated in the table (10th week after sowing) Different letters symbolize statistically significant difference between means according to non-parametric Kruskal–Wallis one-way analysis of variance (p < 0.0001) followed by Dunn’s multiple comparisons test (p < 0.01) The error bars show the standard error of the mean values (n = 300–400) No less importantly, this step was also an attempt to show a correlation between increased levels of fertilization and the size of fungal nutrient deposits (intraradical vesicle number, Figure 2A) Such a correlation was apparent for the plant host resulting in the accumulation of mineral compounds and leaf nitrogen index (NBI) we found that only one fertilizer level (0.5xD) caused an increase in vesicle abundance such a narrowed sampling reduced the variability at distant leaf positions associated with N status diagnosis The high compatibility of the middle leaf NBI index with N content changes allowed us to accept its measurements as a reliable indicator of AM-affected N management efficiency It was confirmed at the same time that the 1xD dose aligns the physiological performance of AM and NM plants in the early generative phase of growth Correlation between the Nitrogen Balance Index (NBI) and the nitrogen content of the middle leaves Data was collected for analysis (n = 32) from four fertilization variants (1xD Both nitrogen content (expressed as weight units per leaf dry mass) and NBI measurements were done on the same plants and leaves (L4 + 5 Pearson’s correlation coefficient (value R2) significant at the level of 0.01% (p < 0.0001) Effect of increasing fertilization on nitrogen status (A) and PSII quantum conversion (B) of the middle leaves as indicators of nutritional status of NM and AM plants chlorophyll fluorescence decrease ratio; Fv/Fm maximum quantum yield of PSII in the dark-adapted state The bars represent average values taken from middle leaves (L4 + 5 ear leaf and leaf above) at the time of tasseling (10 weeks after sowing) after 4 weeks fertilization with one of 4 dilutions of maximal (1xD) dose The error bars show the standard error of the mean (n = 80 for NBI Chl and Flv: 4 plants × 2 leaves × 10 sampling points covering upper half of leaf length Asterisks show statistically significant difference between NM and NM means according to t-Student test (∗p < 0.05 Effect of mycorrizae and fertilization level on light-saturated photosynthetic efficiency of the middle leaves Unchanged Rfd indicated that the plants produced PSII centers, operating with full capacity, for each fertilizing level (Figure 4B) We show this parameter because its sensitivity has proved particularly useful for rapid and non-invasive detection of drought stress progress when it comes to the reduction of the PSII quantum conversion capacity Severe drop in root and leaf water potential (Ψ) was not registered until S13 The RWC (data not shown) in roots and leaves on that day (below 40 and 25% data not shown) indicated that the Ψ decrease was caused mainly by dehydration were equally able to restore Ψ and RWC values in both tissues within 2–3 days after rewatering Effect of progressive drought and recovery on leaf nitrogen (A) and water (B) status PSII quantum conversion (C) and light-saturated photosynthetic efficiency (D) chlorophyll fluorescence decrease ratio; Amax Non-mycorrhized (NM) and mycorrhized (AM) plants were evaluated during full hydration (S0) The bars represent average values taken from combined measurements from the middle leaves (L4 + 5 Chl and Flv: 5 plants × 2 leaves × 10 sampling points covering upper half of leaf length; n = 8 for Rfd AM plants overwhelmed this ability of NM counterparts at each leaf position During drought development, the general response of stomatal gas exchange rate [CO2 assimilation (Amax) and water conductance (gs), both measured under saturated light] followed the pattern of fluorimetric and water potential variability in leaves (Figure 5D) After a large drop of those values in both symbiotic variants the mycorrhizal influence was evident only during the rehydration period when stomatal conductance and CO2 fixation capacity of AM plants restored faster To verify if mycorrhizae enhance corn fodder yield and/or nutritive value if highly fertilized pot cultures are subjected to drought the plants were treated with NPK doses specific to field cultivation of silage corn (moderate 0.5xD or high 1xD) until the grain-filling stage (12 weeks after seeding) Drought was imposed for 2 weeks at the time of silking which is particularly sensitive Then fertilization was renewed for additional 5 weeks until the half-milkline stage of grain development recommended for silage harvest (BBCH stage 85) The plants were cultivated on the substrate taken from preceding AM or NM cultures (i.e. Contrary to expectations, it turned out that mycorrhizal protection against drought stress was not sufficient to increase the final yield and food value (Supplementary Table 1) differences in growth parameters exceeded 10% (weight of leaves but for each dose the variants AM and NM did not differ significantly in growth parameters The nitrogen index (NBI) for individual leaf positions on the shoot at harvest time was higher in more fertilized plants on average by 40% in NM plants or 70% in AM plants (not shown) differences in leaf NBI between symbiotic variants were not specific and differed by up to 20% in favour of AM or NM plants in three independent cultures (not shown) AM had no effect on organic nutrients of fresh corn as well as the estimated nutritive value of corn silage (Supplementary Table 1) Lower crude fiber content together with higher concentrations of crude protein and ether extract resulted in higher nutritive value no statistically significant interaction between the effect of mycorrhiza and fertilization was observed One of the most striking findings of this study was the preservation of high vitality of intraradical hyphae until corn maturity under fertilization regime approximating the nutrient recommendations for field cultivation of silage corn (see Figures 2A,C, and calculations in Supplementary Data Sheet 1 “Hydroponic vs Comparing our semi-hydroponic pot nutrition (expressed in weekly weight doses) with per plant NPK fertilization program for low-fertility soils the only limitation was found in P application reduced to 60% considering medium soil fertility prevailing in Poland the Pi dosage needed to gain moderate corn yield seems to be very close to those we found as not detrimental for mycorrhizal mycelium development irregularis might be undisturbed under fertilization levels recommended in modern crop cultivation The cultures supplied with 1xD fertilizer solution were set up for further evaluation of fungal drought potential in order to minimize interference from nutrient limitation both caused by a reduced use of carbon for synthesis of nitrogen compounds chlorophyll (B) and flavonoids (C) level and leaf area (D) at 8 leaf positions after 2-week drought imposed at silking time (10 weeks after sowing fertilized with 1xD dose) and 7 days of rehydratation The error bars show the standard error of the mean (n = 60 for NBI Chl and Flv: 6 plants × 10 sampling points covering upper half of leaf length Asterisks show statistically significant difference between NM and AM means according to t-Student test (∗p < 0.05 The aim of our second experiment was to observe if progressive drought stress would alter leaf physiology of such a mutant It is plausible that such enhancement of root and leaf water flux might exert a temporary negative effect on leaf physiology and explain our observation of AM-accelerated senescence rate during drought development in field conditions such AM-dependent depletion of nutrients would not be expected since the fungal mycelium system could penetrate the soil unlimited by pot volume Augé et al. (2015) meta-analysis shows that AM alteration of stomatal conductance can occur even if AM and NM plants do not differ in size or tissue P concentration Therefore the notion that AM effects on plant water relations were mainly nutritional in nature the cited authors conclude that researchers can expect to see more mycorrhizal effects on leaf water management when experiments are conducted in nutritionally deficient conditions Our results show that even if corn cultures are grown under non-limiting fertilization the drought-induced senescence and stomatal closure can be altered by hyphal activity in a way apparently not mediated by AM-improved growth more advantageous mycorrhizal effects on crop quantity and quality can be expected either under lower soil nutritional value or when plants are exposed to a much longer period of water shortage All or some of these circumstances might be abolished by mycorrhiza-related growth response in terms of plant biomass or mineral nutrient transfer. It should be noted, however, that neutral mycorrhizal outcome does not unequivocally indicate a truly commensal or parasitic relationship (Smith and Smith, 2011) Even if mycorrhiza does not affect overall growth or mineral uptake by the plant the mycelium – if present in the root cortex – still has a potential to provide an alternative route for water and nutrition supply Such a hidden but mutualistic relationship may show up under abiotic stress situation Improved plant nutrition is the often suggested explanation of mycorrhizal effects on plant productivity and resulting enhancement of drought tolerance This study shows that long-term corn symbiosis with Rhizophagus irregularis might be undisturbed under fertilization levels recommended for field cultivation of silage corn but without any significant fungal effect on fodder yield and quality the reduced disproportion of nutritional status of AM and NM counterparts did not eliminate symbiotic benefits under challenging drought conditions It was particularly remarkable at the time of rewatering when mycorrhiza enhanced plant ability to reverse leaf senescence symptoms These findings are particularly interesting because the corn variety chosen for our study (‘Opoka’) is a stay-green hybrid Such a phenotype is characterized by a lowered rate of developmental senescence during grain filling but additionally associated with enhanced drought resistance severe drought development in our study limited the effectiveness of this mechanism and both AM and NM plants were not able to avoid dehydration We can therefore conclude that genetically based timing and progression of drought-induced senescence could undergo alteration by hyphal activity even in stay-green mutants in a way apparently not mediated by AM-improved growth TL helped design conditions for pot cultures and analyzed the data EP-L was supported by Adam Mickiewicz University Faculty of Biology Dean’s grant no GDWB-07/2014 RM was supported by the statutory 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