Transcriptomic and hormonal analysis of the roots of maize seedlings grown hydroponically at low temperature.

Prolonged cold stress has a strong effect on plant growth and development, especially in subtropical crops such as maize. Soil temperature limits primary root elongation, mainly during early seedling establishment. However, little is known about how moderate temperature fluctuations affect root growth at the molecular and physiological levels. We have studied root tips of young maize seedlings grown hydroponically at 30 ºC and after a short period (up to 24 h) of moderate cooling (20 ºC). We found that both cell division and cell elongation in the root apical meristem are affected by temperature. Time-course analyses of hormonal and transcriptomic profiles were achieved after temperature reduction from 30 ºC to 20 ºC. Our results highlighted a complex regulation of endogenous pathways leading to adaptive root responses to moderate cooling conditions.

Auxin-Cytokinin Balance Shapes Maize Root Architecture by Controlling Primary Root Elongation and Lateral Root Development.

The root system is responsible for water and nutrients uptake from the soil, and therefore, its extension is basic for an efficient acquisition. The maize root system is formed by different types of roots, and the lateral root branching substantially increases the surface for nutrient uptake. Therefore, the regulation of lateral root formation is fundamental in the development of root functions. Root architecture is basically controlled by auxin and cytokinins, which antagonize in the formation of lateral roots (LR) along the primary root axis, with auxin, a stimulator, and cytokinins inhibitors of LR development. This interaction has been analyzed in several zones along the primary root where LRs in different developmental stages were located. The root has been divided into several zones, such as meristem, elongation zone, and mature zone, according to the developmental processes occurring in each one. As Arabidopsis root elongated more slowly than maize root, these zones are shorter, and its delimitation is more difficult. However, these zones have previously been delimitated clearly in maize, and therefore, they analyze the effect of exogenous hormones in several LR developmental stages. The inhibitory effect of cytokinin on lateral root formation was observed in already elongated primary root zones in which initial events to form new lateral roots are taking place. Contrarily, auxin increased LR formation in the primary root segments elongated in the presence of the hormone. The inhibitory effect of cytokinin was reversed by auxin in a concentration-dependent manner when both hormones were combined. However, auxin is unable to recover LR development in primary root zones that have been previously elongated only in the presence of cytokinin. This antagonistic auxin-cytokinin effect on LR development depended on the balance between both hormones, which controls the root system architecture and determines the formation of LR during the process of initiation.

Auxin Modulated Initiation of Lateral Roots Is Linked to Pericycle Cell Length in Maize.

Auxin is essential for the regulation of root system architecture by controlling primary root elongation and lateral root (LR) formation. Exogenous auxin has been reported to inhibit primary root elongation and promote the formation of LRs. In this study, LR formation in the Zea mays primary root was quantitatively evaluated after exogenous auxin treatment by comparing the effects of auxin on two selected zones elongated either before or after auxin application. We determined two main variables in both zones: the LR density per unit of root length (LRD), and the mean phloem pericycle cell length. The total number of phloem pericycle cells (PPCs) per unit of root length was then calculated. Considering that each LR primordium is initiated from four founder cells (FCs), the percentage of PPCs (%PPC) that behave as FCs in a specific root zone was estimated by dividing the number of pericycle cells by four times the LRD. This index was utilized to describe LR initiation. Root zones elongated in the presence of a synthetic auxin (1-naphthalene acetic acid, NAA) at low concentrations (0.01 µM) showed reduced cell length and increased LRD. However, a high concentration of NAA (0.1 µM) strongly reduced both cell length and LRD. In contrast, both low and high levels of NAA stimulated LRD in zones elongated before auxin application. Analysis of the percentage of FCs in the phloem pericycle in zones elongated in the presence or absence of NAA showed that low concentrations of NAA increased the %PFC, indicating that LR initiation is promoted at new sites; however, high concentrations of NAA elicited a considerable reduction in this variable in zones developed in the presence of auxin. As these zones are composed of short pericycle cells, we propose that short pericycle cells are incapable to participate in LR primordium initiation and that auxin modulated initiation of LRs is linked to pericycle cell length.

Transition zone cells reach G2 phase before initiating elongation in maize root apex.

Root elongation requires cell divisions in the meristematic zone and cell elongation in the elongation zone. The boundary between dividing and elongating cells is called the transition zone. In the meristem zone, initial cells are continuously dividing, but on the basal side of the meristem cells exit the meristem through the transition zone and enter in the elongation zone, where they stop division and rapidly elongate. Throughout this journey cells are accompanied by changes in cell cycle progression. Flow cytometry analysis showed that meristematic cells are in cycle, but exit when they enter the elongation zone. In addition, the percentage of cells in G2 phase (4C) strongly increased from the meristem to the elongation zone. However, we did not observe remarkable changes in the percentage of cells in cell cycle phases along the entire elongation zone. These results suggest that meristematic cells in maize root apex stop the cell cycle in G2 phase after leaving the meristem.

The initiation of lateral roots in the primary roots of maize (Zea mays L.) implies a reactivation of cell proliferation in a group of founder pericycle cells.

The initiation of lateral roots (LRs) has generally been viewed as a reactivation of proliferative activity in pericycle cells that are committed to initiate primordia. However, it is also possible that pericycle founder cells that initiate LRs never cease proliferative activity but rather are displaced to the most distal root zones while undertaking successive stages of LR initiation. In this study, we tested these two alternative hypotheses by examining the incorporation of 5-bromo-2'-deoxyuridine (BrdU) into the DNA of meristematic root cells of Zea mays. According to the values for the length of the cell cycle and values for cell displacement along the maize root, our results strongly suggest that pericycle cells that initiate LR primordia ceased proliferative activity upon exiting the meristematic zone. This finding is supported by the existence of a root zone between 4 and 20mm from the root cap junction, in which neither mitotic cells nor labelled nuclei were observed in phloem pericycle cells.

Synergistic action of auxin and ethylene on root elongation inhibition is caused by a reduction of epidermal cell length.

Auxin and ethylene have been largely reported to reduce root elongation in maize primary root. However the effects of auxin are greater than those caused by ethylene. Although auxin stimulates ethylene biosynthesis through the specific increase of ACC synthase, the auxin inhibitory effect on root elongation is not mediated by the auxin-induced increase of ethylene production. Recently it has been demonstrated that root inhibition by the application of the synthetic auxin NAA (1-naphtalenacetic acid) is increased if combined with the ethylene precursor ACC (1-aminocyclopropane-1-carboxilic acid) when both compounds are applied at very low concentrations.   Root elongation is basically the result of two processes: a) cell divisions in the meristem where meristematic cells continuously generate new cells and b) subsequently polarized growth by elongation along the root axis as cells leave the meristem and enter the root elongation zone. Our results indicate that exogenous auxin reduced both root elongation and epidermal cell length. In a different way, ethylene at very low concentrations only inhibited root elongation without affecting significantly epidermal cell length. However, these concentrations of ethylene increased the inhibitory effect of auxin on root elongation and cell length. Consequently the results support the hypothesis that ethylene acts synergistically with auxin in the regulation of root elongation and that inhibition by both hormones is due, at least partially, to the reduction of cell length in the epidermal layer.

Effects of antagonists and inhibitors of ethylene biosynthesis on maize root elongation.

During the first days of development, maize roots showed considerable variation in the production of ethylene and the rate of elongation. As endogenous ethylene increases, root elongation decreases. When these roots are treated with the precursor of ethylene aminocyclopropane- 1-carboxylic acid (ACC), or inhibitors of ethylene biosynthesis 2-aminoethoxyvinyl glycine (AVG) or cobalt ions, the root elongation is also inhibited. Because of root growth diminishes at high or reduced endogenous ethylene concentrations, it appears that this phytohormone must be maintained in a range of concentrations to support normal root growth. In spite of its known role as inhibitor of ethylene action, silver thiosulphate (STS) does not change significantly the root elongation rate. This suggests that the action of ethylene on root elongation should occur, at least partially, by interaction with other growth regulators.