Ectopic Lateral Root Branching in Fe-Deprived Maize Plants: Searching for the Genes Underpinning the Phenotype ^†

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. 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.

		Control			-Fe
Days	PR	SR	CR	PR	SR	CR
7	11.23 ± 2.59	8.77 ± 1.84	0.00	11.23 ± 2.59	8.77 ± 1.84	0.00
14	33.27 ± 5.12	34.70 ± 4.63	15.27 ± 2.15	29.07 ± 4.71	23.17 ± 3.28 *	10.67 ± 1.25 *
21	60.33 ± 8.48	56.57 ± 6.91	38.67 ± 5.33	33.53 ± 5.79 *	22.47 ± 4.35 *	14.47 ± 2.55 *

). 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.

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. 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.

		Control			-Fe
Days	PR	SR	CR	PR	SR	CR
7	40.36 ± 5.18	0.00	0.00	40.36 ± 5.18	0.00	0.00
14	80.46 ± 4.85	77.43 ± 3.26	44.32 ± 4.72	85.09 ± 4.92	83.74 ± 6.77	66.25 ± 5.39 *
21	86.52 ± 3.59	82.14 ± 4.28	77.67 ± 5.63	96.72 ± 3.27 *	93.62 ± 4.30 *	93.55 ± 5.66 *

). Fe deprivation resulted in greater coverage with lateral roots, which was above 90% in all root types on day 21.

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. 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.

		Control			-Fe
Days	PR	SR	CR	PR	SR	CR
7	6.70 ± 0.94	0.00	0.00	6.70 ± 0.94	0.00	0.00
14	6.50 ± 0.91	7.83 ± 0.77	8.50 ± 0.74	4.33 ± 0.57 *	3.77 ± 0.61 *	3.60 ± 0.44 *
21	8.20 ± 1.03	10.10 ± 1.21	7.77 ± 1.16	1.10 ± 0.28 *	1.03 ± 0.14 *	0.77 ± 0.18 *

, 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.

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. List of the genes in the QTL located in chromosome 1, between the flanking markers “chrom7-glb1” (V4 annotation of MGDB).

MGDB Gene ID	MGDB Gene/Protein Information
Zm00001d033374	TIDP3143, ubiquitin-conjugating enzyme
Zm00001d033375	cl27571_1, FMP27, GFWDK domain protein
Zm00001d022967	lincRNA
Zm00001d033377	9-cis-epoxycarotenoid dioxygenase (abscisic acid biosynthesis)
Zm00001d022968	lincRNA
Zm00001d033378	hb33, homeobox-transcription factor 33
Zm00001d033379	kip1, knotted interacting protein1 transcription factor
Zm00001d033380	tetratricopeptide-like helical domain superfamily protein
Zm00001d033382	-
Zm00001d033383	thic1, hydroxymethylpyrimidine phosphate synthase1 (thiamine biosynthesis)
Zm00001d033384	ribosomal protein L5 domain family
Zm00001d033385	UDP-d-galacturonate:1,4-a-poly-d-galacturonate 4-a-d-galacturonosyltransferase (homogalacturonan biosynthesis)
Zm00001d033386	zinc finger, RING/FYVE/PHD-type domain containing protein
Zm00001d033388	tetratricopeptide-like helical domain superfamily protein
Zm00001d033389	vq10, VQ motif-transcription factor10
Zm00001d022969	lincRNA
Zm00001d033390	basic-leucine zipper domain transcription factor
Zm00001d033391	-
Zm00001d033392	-
Zm00001d033393	-
Zm00001d033394	-
Zm00001d033395	-
Zm00001d022970	lincRNA
Zm00001d033396	grftf4, Growth-Regulating Factor (GRF)—transcription factor 4
Zm00001d033397	cl9255_1, CNNM transmembrane domain protein
Zm00001d033399	-
Zm00001d033400	metal-dependent protein hydrolase domain
Zm00001d033401	osca7, hyperosmolality-gated calcium-permeable channels7
Zm00001d033402	-
Zm00001d033403	tRNA
Zm00001d033404	hb110, homeobox-transcription factor 110
Zm00001d033405	TIDP3692, pyruvate kinase

. 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.

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.