Abstract
Iron (Fe) is an essential element for plant growth and productivity, and human and animal diets rely on Fe from plant sources. Despite the large number of studies on plants’ Fe deficiency responses, considerably less is known about the morphological and anatomical alterations that root systems of plants undergo, especially in the graminaceous plants following a chelation strategy to take up Fe3+ from the rhizosphere. A stress symptom observed in Fe-deprived maize plants is an ectopic lateral root branching occurring at the terminal 5 cm of the root. In order to understand this response, one-week-old maize seedlings were placed in containers with either full nutrient solution, or nutrient solution lacking an Fe source. Control and Fe-deprived plants were grown for another 14 days, and the trait of ectopic lateral root branching was observed both on roots that emerged before the onset of Fe deprivation, as well as on roots that emerged after the onset of the deprivation. Ongoing in silico analysis of a quantitative trait locus known to be related to this trait of maize grown under limited Fe, revealed several genes coding for known and unknown proteins, as well as long intergenic non-coding RNAs.
1. Introduction
In the soil, Fe is found in the form of Fe3+ (ferric iron) and Fe2+ (ferrous iron) and is highly dependent on the redox state of its environment. Organisms have developed specific mechanisms for Fe acquisition from the Fe-oxides (III): (i) protonation, (ii) chelation, and (iii) reduction [1].
Eudicots and non-graminaceous monocots employ Strategy I to acquire iron from the soil. In this strategy, also known as reduction strategy, protons are pumped to the rhizosphere by activation of a plasma membrane H+-ATPase, which promotes the acidification of the soil solution and increases solubility of Fe3+ [2]. Subsequently, the ferric reductase enzyme, encoded by the ferric chelate reductase gene, reduces Fe3+ to Fe2+ in the surface of the plasma membrane of root cells. After reduction, Fe is transported to the cytoplasm through iron-regulated transporters. Additionally, Strategy I plants were found to export an array of metabolites including organic acids, phenolics, flavonoids, and flavins [3].
In Strategy II, also referred as chelation strategy, which is typical of the grass family (Poaceae), the roots release compounds called phytosiderophores. These molecules belong to the mugineic acid family (MAs) and can form stable complexes with Fe3+ in the rhizosphere. Deoxymugineic acid is the most abundant phytosiderophore and is exported by TOM1, the transporter of mugineic-acid-family phytosiderophores, in rice and barley [4]. The absorption of Fe3+-phytosiderophore complexes is performed by a yellow stripe/yellow-stripe-like transporter located in plant root cells. It is well established that maize (Zea mays L.) follows this strategy for iron acquisition [5].
Iron is an essential element for plant growth and productivity, and it is necessary to understand the responses of plants to iron deficiency at both the physiological and morphological levels and reveal the molecular and genetic bases of these responses. Although there is a large number of studies available on plants’ iron deficiency responses, satisfactory knowledge regarding the morphological and anatomical alterations in plant root systems is lacking, especially in the graminaceous plants. It seems that plants modify their root architecture by increased formation and branching of root hairs, root-tip swelling, and enhanced lateral root formation [6,7]. Especially for maize, a stress symptom observed in Fe-deprived plants is an ectopic lateral root branching at the terminal 5 cm of the root. In this study, control and Fe-deprived plants were grown for 3 weeks, and the trait of ectopic lateral root branching was observed both on roots that emerged before the onset of Fe deprivation (i.e., primary embryonic and secondary embryonic roots), as well as on roots which developed after the onset of the deprivation (i.e., crown roots). Bioinformatic analysis of a quantitative trait locus known to be related to this trait of maize grown under limited Fe [6], resulted in a list of several genes coding for known and unknown proteins, with many transcription factors among them, as well as long intergenic non-coding RNAs.
2. Methods
2.1. Plant Growth Conditions and Treatments
Maize seeds (Zea mays L.) were placed on wet filter paper, in the dark for germination. Four days later, the most uniform plants were selected and maintained in a hydroponic batch culture for 3 days in well-aerated distilled water. On day 7 after sowing and for the next week they were transferred in a hydroponic batch culture, and separated into two different treatments: in the first one, plants were set to grow in complete nutrient solution (C, 5 mM KNO3, 1 mM KH2PO4, 2 mM Mg(NO3)2 6H2O, 2.5 mM CaSO4 2H2O, 1 mM MgSO4 7H2O, 0.07 mM FeNaEDTA, 4 mM Ca(NO3)2 4H2O, 0.9 μM ZnCl2, 30 μM H3BO3, 0.9 μM CuCl2, 0.5 μM MoO3 85%, and 20 μM MnCl2 4H2O) while on the other, plants were subjected to iron-deficient conditions (-Fe, 5 mM KNO3, 1 mM KH2PO4, 2 mM Mg(NO3)2 6H2O, 2.5 mM CaSO4 2H2O, 1 mM MgSO4 7H2O, 4 mM Ca(NO3)2 4H2O, 0.9 μM ZnCl2, 30 μM H3BO3, 0.9 μM CuCl2, 0.5 μM MoO3 85%, and 20 μM MnCl2 4H2O). All nutrient solutions were constantly aerated and replaced every 3 days, throughout the experiment. Growth conditions were 24/18 °C, relative humidity 40%, 250 µmol photon m−2 s−1 and a 16-h photoperiod.
2.2. Samplings and Observations
Each experiment was repeated twice. Samplings (five replicates per sampling) took place on day 7 prior to separation into the two nutrient solutions, as well as on days 14 and 21 after sowing. Root length, the percentage of root length covered with lateral roots, and the distance between the root tip and the point of lateral root emergence were measured in primary embryonic (PR), seminal embryonic (SR), and crown (CR) roots on days 7, 14, and 21 after sowing.
2.3. In Silico Analysis
Bioinformatic analysis was performed using the data from the Maize Genetics and Genomic Database (MGDB): https://www.maizegdb.org/ (accessed date: 1 November 2020) [8].
2.4. Statistical Analysis
In order to determine the significance of differences between samplings, the data were analyzed using the t-test variance analysis with two-tailed distribution and two-sample unequal variance in Microsoft Excel.
3. Results and Discussion
In Fe-deprived plants, the length of all root types examined was reduced compared to controls during the treatment. More specifically, the length of the primary root (PR) was reduced on day 21, whilst the respective lengths of the seminal (SR) as well as the crown (CR) roots were significantly decreased from day 14 onwards (Table 1). It is worth noting that on day 21, the roots of the Fe-deprived plants were almost half the length of these in control plants.
Table 1.
Root length (cm, mean ± SE) of primary (PR), seminal (SR), and crown (CR) roots on days 7, 14, and 21 after sowing. Asterisk (*) indicates statistically significant difference between -Fe treatment and the respective control at p < 0.05.
The observation of the root area covered with lateral roots revealed the presence of statistically significant differences between control and Fe-deprived roots both on days 14 and 21, in the case of CR, and on day 21 for PR and SR (Table 2). Fe deprivation resulted in greater coverage with lateral roots, which was above 90% in all root types on day 21.
Table 2.
Percentage (%, mean ± SE) of root length of primary (PR), seminal (SR), and crown (CR) roots covered with lateral roots on days 7, 14, and 21 after sowing. Asterisk (*) indicates statistically significant difference between -Fe treatment and the respective control at p < 0.05.
The above-mentioned increase in the area covered with lateral roots under Fe-deprived conditions can be attributed to the corresponding decrease of the distance between the root tip and the point of lateral root initiation. As shown in Table 3, the presence of lateral roots was observed at a distance shorter than 5 cm from the root tip from day 14 onwards and on all maize root types. On day 21, emerging lateral roots were observed at a distance of 1 cm from the root tip.
Table 3.
Distance (cm, mean ± SE) of the point of lateral root emergence and root tip in primary (PR), seminal (SR), and crown (CR) roots on days 7, 14, and 21 after sowing. Asterisk (*) indicates statistically significant difference between -Fe treatment and the respective control at p < 0.05.
The study of the quantitative trait loci (QTLs) associated with the formation of lateral roots at a distance shorter than 5 cm from the root tip under Fe deprivation (branching at the terminal 5 cm of root, BTR), revealed three QTLs [6]. Among these, we chose to study the QTL located in chromosome 1, between the flanking markers “chrom7-glb1”, primarily due to the relatively high percentage of the explained phenotypic variance (20.9%) by this QTL. Interestingly, the same QTL is also responsible for the variation of the SPAD value of younger leaves. The genes found in chrom7-glb1 are listed in Table 4. Among these four genes coding for long intergenic non-coding RNAs (lincRNAs), six genes coding for transcription factors, as well as eight genes coding for unknown proteins, are present.
Table 4.
List of the genes in the QTL located in chromosome 1, between the flanking markers “chrom7-glb1” (V4 annotation of MGDB).
Among the genes contained into this region of the maize genome, there are three genes related to hormonal biosynthesis and/or signaling. The gene Zm00001d033377 codes for an enzyme of the abscisic acid biosynthetic pathway, Zm00001d033375 codes for an enzyme related to ethylene response, whilst Zm00001d033379 and Zm00001d033396 code for two transcription factors related to gibberellin signaling (regulation of gibberellin biosynthesis and response to gibberellin, respectively). Additionally, Zm00001d033375 is also related to cellular response to phosphate starvation. The gene Zm00001d033383 codes for an Fe–S cluster enzyme involved in thiamin biosynthesis. Future research is required in order to reveal the underlying mechanisms by which the BTR phenotype occurs when plants experience Fe starvation as well as how this phenotype improves Fe homeostasis in maize plants.
4. Conclusions
The trait of lateral root branching at the terminal 5 cm of the root in Fe-deprived maize plants was observed both in roots that emerged before the onset of Fe deprivation (i.e., the primary embryonic root and the secondary embryonic ones), as well as on roots that emerged after the onset of the deprivation (i.e., the crown roots). Bioinformatic analysis resulted in the identification of a region in maize genome chromosome underpinning this phenotype.
Author Contributions
S.N.C. and Y.E.V. conceived and designed the experiments; Y.E.V., S.-T.P.P., and A.-E.N. performed the experiments; S.N.C. and Y.E.V. analyzed the data; S.N.C., Y.E.V., and D.L.B. wrote the paper. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions.
Conflicts of Interest
The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
| PR | Primary embryonic roots |
| SR | Seminal embryonic roots |
| CR | Crown roots |
| BTR | Branching at the terminal 5 cm of root |
| lincRNA | Long intergenic non-coding RNA |
| MGDB | Maize Genetics and Genomic Database |
| QTL | Quantitative trait locus |
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