1,720,969 research outputs found
NITRATE SENSING BY MAIZE ROOTS: A KEY ROLE FOR NITRIC OXIDE SIGNALING IN THE TRANSITION ZONE
Full text linkOver the past five decades, intensive agriculture has been able to increase the rate of food production more rapidly than that of human population growth but, at the same time, has also given rise to a series of negative environmental consequences, at both local and global level. Most of them are directly due to the large use in agriculture of synthetic ammonia fertilizers industrially produced by the Haber-Bosch process. Over 50% of the applied nitrogen in fact, is lost from the plant-soil system, leading to severe environmental damages and to negative impacts on human health. Maize (Zea mays L.) is one of the world’s major crops and is also expected to give an important contribution to human nutrition in the next few decades, when world population should exceed 8 billion people and rise to 9.2/11 billion by 2050. To ensure future global food security, increasing crop yields are dramatically needed, however, sustainable ways of crop production are far from being achieved, considering also that further nitrogen accumulation in the environment is expected to be increased in the future without an adequate enhancement of Nitrogen Use Efficiency (NUE) in the main crops. For this reason, the understanding of the molecular events underlying root adaptation to nitrogen fluctuations is a primary goal to develop tools for sustainable agriculture.
Crop plant development in fact, is not only strongly dependent on nitrogen availability in the soil but also on the efficiency of its recruitment by roots. Plants take up and assimilate both nitrate and ammonium, but nitrate is the main source of inorganic nitrogen for plants in aerobic soil conditions typical of most cultivated soils. In addition to its role as a nutrient, nitrate acts as a signaling molecule regulating the expression of the genes involved in growth and developmental processes. However, the mechanisms governing the sensing of nitrate by roots and of the signaling leading to an altered development of roots are still only partially characterized. Nitric oxide (NO) has been recently proposed to be implicated in plant responses to environmental stresses, but its exact role in the response of plants to nutritional stress is still under evaluation. In this work, the role of NO production by maize roots after nitrate perception was investigated by focusing on the regulation of transcription of genes involved in NO homeostasis and by measuring NO production in roots. Moreover, its involvement in the root growth response to nitrate was also investigated.
To better discriminate nitrate-specific effects from those more generally N-dependent, the expression of a number of genes previously identified as being nitrogen-responsive, was evaluated in response to nitrate/ammonium supply and deprivation. The transcriptional response of five genes encoding (i) the cytosolic nitrate reductase NR1, (ii) two different non-symbiotic hemoglobins (nsHbs) isoforms, (iii) a gene encoding nitrite reductase together with (iv) a gene encoding the high-affinity root nitrate transporter (NRT2.1), evidenced a very strong and exclusive nitrate responsiveness in roots. Conversely, no effects were observed when ammonium was supplied as the sole nitrogen source. This first screening allowed the current work to focus later only on genes whose expression seems to depend exclusively on nitrate and to be specifically involved in the control of NO biosynthesis and scavenging. Our results highlight the importance of the coordinate spatio-temporal expression of nitrate reductase and non-symbiotic hemoglobins in controlling the NO homeostasis in the maize root after nitrate provision.
In addition, data obtained by analysing root morphological parameters by the WinRhizo software underlined the same specificity of nitrate, which significantly affected root growth when supplied to N-deprived roots.
To deepen the hypothesis that nitric oxide may be produced by roots as an early signal of nitrate perception, NO in vivo detection was carried out. Results obtained using the DAF-2DA probe and stereo- and confocal microscopy evidenced a clear induction of fluorescence after nitrate provision. Very interestingly, the main zone of NO production seemed to be located immediately above the meristematic apex and more precisely to coincide with the root transition zone. The fluorescence detected after nitrate supply was not revealed in the presence of the specific nitrate reductase inhibitor tungstate, giving support to the role of NR in nitric oxide production. Moreover, the addition of the nitric oxide scavengers cPTIO together with nitrate, similarly suppressed the development of fluorescence, confirming the specificity of NO detection by the probe. These results suggest that a NR-dependent NO burst occurred immediately after nitrate supply to roots. The NR-dependent NO production observed after nitrate supply was then further confirmed by the strong induction of NR1, NiR, and nsHbs transcription in the early phases of nitrate perception. In this case also, the transcription was significantly inhibited in response to tungstate and cPTIO addition, endorsing the cooperation between nitrate reductase and haemoglobin activities in the finely tuned control of NO homeostasis.
To deepen the spatial regulation of NO homeostasis balance, the expression of NR1, NiR and nsHbs genes was also analysed in four different root zones (i.e. meristem, transition zone, elongation zone, maturation zone) both in nitrate-depleted and in nitrate-treated seedlings. In N-starved roots, all transcripts evidenced their maximum accumulation at the meristem level. This pattern radically changed when nitrate was furnished to roots with a very significant increase of transcript abundance in the transition zone. As a result, we suggest that nitrate supply could activate its own sensing by stimulating NO production by the transition zone cells, thus initiating a signalling pathway contributing to the physiological adaptation (e.g. root growth) to nitrate fluctuations.
Based on the preliminary results showing the preferential localization of NO production at the level of the transition zone, the attention was then focused on nitrate effects on root elongation, which takes place in the zone immediately above and neighbouring the transition zone. Our finding evidenced a strong and specific induction of root elongation of young maize seedlings supplied with 1 mM nitrate and a drastic inhibition in the presence of ammonium, cPTIO, and tungstate. On the contrary, when the negative control (-NO3-) was supplied with a NO donor (SNP) the root length increased significantly. These results strongly suggest that the NO generated through NR should significantly contribute to the root lengthening noticed after nitrate provision.
To summarize, it would seem that the NO-mediated pathway here described represents an early alert system for external nitrate sensing by root cells, which seem to individually possess the competence to activate this pathway when external nitrate is perceived. Additional experiments are necessary to better understand the functioning of this NO-mediated pathway and to identify the downstream events that link the NO burst with the physiological redirection of root growth. In this regard, it has been reported that NO signaling can alter cell polarity and cytoskeleton-mediated vesicle trafficking processes, thus affecting cell growth and root morphogenesis. This suggests that there should be more downstream effectors of NO action, acting either in parallel or in series with cytoskeletal constituents. Furthermore, since NO and phytohormones auxin act synergically to control diverse aspects of root biology and also considering that lateral root development in response to nitrate is strongly auxin dependent, a role of NO as a coordinator of nitrate and auxin signaling to control the overall root response to the anion cannot be excluded.
In order to try to answer to these last questions, in the final part of my Ph. D. thesis, we focused the attention on studying both cytoskeleton-mediated xyloglucans (a major primary cell wall component) modifications and polar auxin transport in the maize root transition zone cells in response to nitrate. Preliminary data achieved so far by using immunofluorescence labelling indicate that nitrate is able to modify cell wall recycling in the transition zone. Xyloglucans in fact, were very abundant especially in the sample subjected to nitrate treatment, when compared to the negative control, suggesting a higher rate of XGs synthesis /or recycling, in response to the anion in the maize root transition zone. Additionally, Brefeldin A (a chemical which prevents vesicle formation in the exocytosis pathway while allowing endocytosis, resulting in the cytoplasmic accumulation of all recycling molecules) treatment partially failed in removing all XGs from cell walls in +N samples, since a marked immunofluorescence was still visible at cross walls, despite the strong effect of the drug that resulted in the abundance of BFA-compartments also within these cells. These latter data could suggest that nitrate promotes a higher rate of XGs recycling in order to maintain a loosened cell wall structure, thus allowing an extensive and fast cell elongation in response to the anion. Taken together, these data open a fascinating scenario in which nitrate might act in promoting rapid cell elongation of root apex by regulating, in a mechanism as yet unknown, the synthesis or the turn-over (or both) of xyloglucans within root transition cells. Also PIN1-mediated auxin accumulation seems to be interfered in response to nitrate. IAA signal in fact, was strongly localized at the cross wall (end-poles) of transition zone cells only in nitrate-supplied roots, thus suggesting that IAA end-poles labelling was probably due to increased IAA fluxes triggered specifically by nitrate. In support to this hypothesis we also observed that IAA and its transporter PIN1 protein co-localize in NO3--treated roots at the cross walls (end-poles), thus providing further, although preliminary, evidences that nitrate in the maize root transition zone is able to increase IAA-fluxes, in a mechanism as yet unknown, that involved also PIN1 proteins. Further immunolabeling data, by also using NO donors and scavengers, will be needed to better understand the coordinated actions of nitric oxide, auxin and cytoskeleton adjustments in tightly regulating root motoric response to nitrate
NO signaling is a key component of the root growth response to nitrate inZea maysL.
Full text linkRoots are considered to be a vital organ system of plants
due to their involvement in water and nutrient uptake, anchorage,
propagation, storage functions, secondary metabolite
(including hormones) biosynthesis, and accumulation. Crops
are strongly dependent on the availability of nitrogen in
soil and on the efficiency of nitrogen utilization for biomass
production and yield. However, knowledge about molecular
responses to nitrogen fluctuations mainly derives from the
study of model species. Nitric oxide (NO) has been proposed
to be implicated in plant adaptation to environment, but its
exact role in the response of plants to nutritional stress is still
under evaluation. Recently a novel role for NO production and
scavenging, thanks to the coordinate spatio-temporal expression
of nitrate reductase and non-symbiotic hemoglobins, in
the maize root response to nitrate, has been postulated. This
control of NO homeostasis is preferentially accomplished by
the cells of the root transition zone (TZ) which seems to represent
the most nitrate responsive portion of maize root. The TZ
is already known to function as a sensory center able to gather
information from the external environment and to re-elaborate
them in an adequate response. These results indicate that
it could play a central role also for nitrate sensing by roots. A
lot of work is still needed to identify and characterize other
upstream and downstream signals involved in the “nitrate-
NO” pathway, leading to root architecture adjustments and
finally to stress adaptation
Identification and characterization of the BZR transcription factor family and its expression in response to abiotic stresses in Zea mays L.
Full text linkBrassinosteroids (BRs) are plant specific steroidal hormones that play diverse roles in regulating a broad spectrum of plant growth and developmental processes, as well as, in responding to various biotic and abiotic stresses. Extensive research over the years has established stress-impact-mitigating role of BRs and associated compounds in different plants exposed to various abiotic and biotic stresses, suggesting the idea that they may act as immunomodulators, thus opening new approaches for plant resistance against hazardous environmental conditions. In this research the characterization of the transcriptional response of 11 transcription factors (TFs) belonging to BRASSINAZOLE-RESISTANT 1 (BZR1) TF family of Zea mays L. was analyzed in seedlings subjected to different stress conditions. Being important regulators of the BR synthesis, BZR TFs might have stress resistance related activities. However, no stress resistance related functional study of BZR TFs has been reported in maize so far. In silico analyses of the selected 11 TFs validated the features of their protein domains, where a highest degree of similarity observed with recognized BZR TFs of rice and Sorghum bicolor. Additionally, we investigated the organ-specific expression of 11 ZmBZR in maize seedlings. Five of them did not show any transcript accumulation, suggesting that ZmBZR expression might be regulated in a manner dependent on plant developmental stage. For the remaining six ZmBZR, their ubiquitous expression in the whole plant indicates they could function as growth regulators during maize development. More importantly, in response to various stress conditions, the spatial transcript accumulation of all ZmBZR varies along the plant. All six ZmBZR showed up-regulation against N starvation, hypoxia and salt stress. On the contrary, heat stress clearly down-regulated gene expression of all ZmBZR analysed. Consistently with the expression results, the distribution of stress-related cis-acting elements in the promoter of these genes inferred that the maize BZR TFs might play some roles in regulating the expression of the corresponding genes in response to multifarious stresses. In conclusion, these data reveal that BZR TFs have stress signaling activity in maize, in addition to their confirmed role in regulating plant physiology and morphology
Evaluation of candidate reference genes for qPCR in maize
No full textQuantitative real-time PCR (qPCR) is a powerful tool to measure gene expression levels. Accurate and
reproducible results are dependent on the correct choice of the reference genes for data normalization.
To date, screenings evaluating candidate reference gene stability for expression studies in maize have
not been reported. In the present work, we analyzed the expression patterns of 12 genes in a set of 20
maize samples, obtained from different tissues of plants grown at various experimental conditions. Using
genormPLUS, NormFinder and BestKeeper algorithms, the expression stability of three “classical” reference
genes, such as ACT, TUB and 18S rRNA, and the newly identified candidates, was assessed. With respect to
the algorithms, our results showed similar performance among genormPLUS, NormFinder and BestKeeper
in evaluating the suitability of reference genes. Our data therefore showed that the currently and widely
used reference genes for data normalization in maize were not the most stable expressed transcripts. Five
of the new putative reference genes (CUL, FPGS, LUG, MEP and UBCP) exhibited the highest expression
stability according to all algorithms. In conclusion, with this study, we provide a list of validated reference
genes and their relative primer sequences to conduct reliable qPCR experiments in maize
A Novel Biostimulant, Belonging to Protein Hydrolysates, Mitigates Abiotic Stress Effects on Maize Seedlings Grown in Hydroponics
Full text linkThe main challenge to agriculture worldwide is feeding a rapidly growing human population, developing more sustainable agricultural practices that do not threaten human and ecosystem health. An innovative solution relies on the use of biostimulants, as a tool to enhance nutrient use efficiency and crop performances under sub-optimal conditions. In this work a novel biostimulant(APR®,ILSAS.p.A.,ArziganoVI,Italy), belongingtothegroupofproteinhydrolysates, wassuppliedtomaizeseedlingsinhydroponicanditseffectswereassessedincontrolconditionsand in the presence of three different kinds of stresses (hypoxia, salt and nutrient deficiency) and of their combination. OurresultsindicatethatAPR® issolubleandisabletoinfluencerootandshootgrowth depending on its concentration. Furthermore, its effectiveness is clearly increased in condition of single or combination of abiotic stresses, thus confirming the previously hypothesised action of this substance as enhancer of the response to environmental adversities. Moreover, it also regulates the transcription of a set of genes involved in nitrate transport and ROS metabolism. Further work will be needed to try to transfer this basic knowledge in field experiments
mRNA-Sequencing Analysis Reveals Transcriptional Changes in Root of Maize Seedlings Treated with Two Increasing Concentrations of a New Biostimulant
Full text linkBiostimulants are a wide range of natural or synthetic products containing substances and/or microorganisms that can stimulate plant processes to improve nutrient uptake, nutrient efficiency, tolerance to abiotic stress, and crop quality ( http://www.biostimulants.eu/ , accessed September 27, 2017). The use of biostimulants is proposed as an advanced solution to face the demand for sustainable agriculture by ensuring optimal crop performances and better resilience to environment changes. The proposed approach is to predict and characterize the function of natural compounds as biostimulants. In this research, plant growth assessments and transcriptomic approaches are combined to investigate and understand the specific mode(s) of action of APR, a new product provided by the ILSA group (Arzignano, Vicenza). Maize seedlings (B73) were kept in a climatic chamber and grown in a solid medium to test the effects of two different combinations of the protein hydrolysate APR (A1 and A1/2). Data on root growth evidenced a significant enhancement of the dry weight of both roots and root/shoot ratio in response to APR. Transcriptomic profiles of lateral roots of maize seedlings treated with two increasing concentrations of APR were studied by mRNA-sequencing analysis (RNA-seq). Pairwise comparisons of the RNA-seq data identified a total of 1006 differentially expressed genes between treated and control plants. The two APR concentrations were demonstrated to affect the expression of genes involved in both common and specific pathways. On the basis of the putative function of the isolated differentially expressed genes, APR has been proposed to enhance plant response to adverse environmental conditions
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Genome-wide discovery and characterization of nitrate-responsive miRNAs in roots of maize seedlings
No full textAbstract
Nitrogen availability affects crops productivity and environment. The natural abundance of useable nitrogen is so low that the massive human alteration of the nitrogen cycle has been required to sustain the feeding of the world's population. Tons of nitrogenous fertilizers are added to the soil worldwide annually, giving rise to environmental diseases. In this scenario, the knowledge of post-transcriptional regulation of plant response to nutrients is important to improve nitrogen use efficiency of crop. With the identification of stress-responsive miRNAs, a layer of post-transcriptional gene regulation has been uncovered.
We used a maize miRNAs-microarray platform to discover previously unknown nitrate-responsive miRNAs. Six mature miRNAs were identified and their expression profiles were studied by quantitative Real Time PCR (qPCR) and in situ hybridization (ISH) in maize roots grown in different nitrate availabilities.
Significant differences in miRNAs’ transcripts accumulation were evidenced between nitrate-supplied and nitrate-depleted roots. Real time PCR analyses and in situ detection of miRNAs confirmed the arrays data and evidenced distinct miRNAs spatiotemporal expression patterns in maize roots.
An in silico approach was used to select target genes of the miRNAs identified. Their transcripts accumulation has been investigated in both nitrate-supplied and nitrate-depleted roots by means of qPCR and ISH.
Our results suggest that miRNAs play some role in modulating N-responsive gene expression by inducing post-transcriptionally the expression of target genes. In particular, the repression of the transcription of miRNA identified upon nitrate shortage could represent a crucial step integrating nitrate signals into developmental changes in maize roots
Nitrate sensing by the maize root apex transition zone: A merged transcriptomic and proteomic survey
Full text linkNitrate is an essential nutrient for plants, and crops depend on its availability for growth and development, but its presence in agricultural soils is far from stable. In order to overcome nitrate fluctuations in soil, plants have developed adaptive mechanisms allowing them to grow despite changes in external nitrate availability. Nitrate can act as both nutrient and signal, regulating global gene expression in plants, and the root tip has been proposed as the sensory organ. A set of genome-wide studies has demonstrated several nitrate-regulated genes in the roots of many plants, although only a few studies have been carried out on distinct root zones. To unravel new details of the transcriptomic and proteomic responses to nitrate availability in a major food crop, a double untargeted approach was conducted on a transition zone-enriched root portion of maize seedlings subjected to differing nitrate supplies. The results highlighted a complex transcriptomic and proteomic reprogramming that occurs in response to nitrate, emphasizing the role of this root zone in sensing and transducing nitrate signal. Our findings indicated a relationship of nitrate with biosynthesis and signalling of several phytohormones, such as auxin, strigolactones, and brassinosteroids. Moreover, the already hypothesized involvement of nitric oxide in the early response to nitrate was confirmed with the use of nitric oxide inhibitors. Our results also suggested that cytoskeleton activation and cell wall modification occurred in response to nitrate provision in the transition zone
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