A role for redox factors in shaping root architecture under phosphorus deficiency

Abstract

The developmental response of the Arabidopsis root system to low phosphorus (P) availability involves the reduction in primary root elongation accompanied by the formation of numerous lateral roots. We studied the roles of selected redox metabolites, namely, radical oxygen species (ROS) and ascorbic acid (ASC) in the regulation of root system architecture by different P availability. Rapidly growing roots of plants grown on P-sufficient medium synthesize ROS in root elongation zone and quiescent centre. We have demonstrated that the arrest of root elongation at low P medium coincides with the disappearance of ROS from the elongation zone. P-starvation resulted in a decrease in ascorbic acid level in roots. This correlated with a decrease in cell division activity. On the other hand, feeding P-deficient plants with ASC, stimulated mitotic activity in the primary root meristem and partly reversed the inhibition of root growth imposed by low P conditions. In this paper, we discuss the idea of the involvement of redox agents in the regulation of root system architecture under low P availability.

Phosphorus (P) Availability Defines the Patterns of Root Architecture in Arabidopsis Affecting Cell Divisions and Elongation

The seedlings of Arabidopsis thaliana develop clearly distinguishable patterns of root system architecture in response to variable P availability. Cultures at low P concentration (1 µM) result in a reduction in primary root growth, increased lateral root formation and enhanced root hair development. On the other hand, at high P concentration (1 mM), the root system is composed of a long primary root with few lateral roots and short root hairs.^1–3

Responses of the root system to P deficiency are dependent on changes in cell proliferation. Arrest of primary root growth at low P availability is due to the inhibition of cell division and the onset of cell differentiation within the primary root meristem. Mitotic activity is relocated to the sites of lateral root formation, which results in an increased lateral root density. In a manner similar to the primary root tip, cell differentiation in older lateral roots occurs within the apical root meristem, which is followed by an arrest in lateral root elongation.^4,5 Besides the reduction in cell division rate, low P treatments inhibit cell growth in root elongation zone.^5,6

Promotion of lateral root development and the arrest of cell divisions in the apices of roots of P-starved plants results from changes in auxin transport and/or sensitivity.^3,4 However, the processes affected by P-deficiency, namely, cell division and elongation are to a large extent, regulated by redox factors like radical oxygen species (ROS) or ascorbate.⁷ Redox agents are involved in an auxindependent patterning in the root apical meristem. Cells of the quiescent centre (QC) that accumulate high auxin levels are characterized by the oxidized status of ascorbate and glutathione and the overproduction of ROS. On the other hand, in rapidly dividing cells of the proximal meristem, the reduced status of ascorbate and glutathione predominates and the ROS levels are low.⁸ It was hypothesized that auxin, by regulating redox status of the discrete zones within the root apical meristem, may act as a positional signal and mediate meristem patterning.⁸

ROS are also involved in the auxindependent regulation of cell elongation. They mediate in the auxin-dependent stimulation of coleoptile growth of maize seedlings.⁹ In contrast, when auxin inhibits maize root growth, there is a decrease in ROS production.¹⁰ Finally, both root hair development and growth, as well as lateral root formation in Arabidopsis root systems, require ROS, produced by AtrbohC NADPH oxidase.¹¹

P Deficiency Affects ROS Distribution in Distal Parts of Arabidopsis Roots

To determine whether P availability affects ROS production, we tested ROS distribution in the apices of the primary roots, lateral root primordia and lateral roots of seedlings grown on media supplemented with a sufficient (1 µM PO₄³⁻) or deficient (1 µM PO₄³⁻) phosphate concentration. As an indicative of ROS levels in discrete segments of roots, 2′,7′-dichlorofluorescein (DCF) fluorescence revealing the patterns of H₂O₂ distribution and nitroblue tetrazolium (NBT) staining indicating the sites of superoxide (O₂^·−) production were analyzed.¹²

Apex of the primary root was reported to have two areas of ROS production: the quiescent centre (QC) and the elongation zone.^8,10 The oxidative environment in the QC is important in maintaining the low cell division rate in the QC.⁸ ROS production in the elongation zone of roots is related to the promotion of the increase in cell wall extensibility within this part of the root.¹⁰ We found that the typical pattern of ROS distribution in the root apex with the local maxima in the QC and elongation zone was visible in roots of seedlings grown in the presence of high (1 mM) P concentration. Contrastingly, when plants were grown on medium with 1 µM P, no distinct fluorescence maximum was detected within the elongation zone, however, DCF and NBT staining maxima were still observed in the QC.¹²

Similar to the primary roots, lateral roots of phosphate sufficient plants accumulated ROS in the elongation zone. In organs belonging to plants grown under low P conditions, ROS were produced in the proximal part of the apical root meristem, while elongation zone remained ROS free.¹²

Our data suggest that the inhibition of cell divisions and the reduction in cell growth in the root elongation zone, which are responsible for the arrest of the primary root growth in P-deficient plants,⁵ are accompanied by an elimination of the ROS distribution pattern typical for growing roots. This includes a decrease in ROS level in the elongation zone (Fig. 1). ROS production in this segment of the root is considered as an important factor that accelerates root growth.¹⁰

Figure 1.

Production of hydrogen peroxide in elongation zone of roots of Arabidopsis seedling cultured 10 days on medium supplemented with 1 µM (A) or 1 mM P (B). Hydrogen peroxide levels were visualized by 2′,7′-dichlorofluorescein (DCF) fluorescence and imaged in an confocal microscope using 488 nm excitation and 525 nm emission spectra. Magnification: 600x. The distribution of DCF fluorescence shows that ROS are synthesized in the cell wall compartment of the elongation zone. Plants grown under P deficiency are characterized by reduced levels of ROS in the cell walls (A) when compared to those cultured on high P concentration (B). Given that ROS-dependent cell wall loosening in a root elongation zone promotes root growth,¹⁰ the decrease in ROS synthesis may be partly responsible for the arrest in root elongation under P deficiency.

An Involvement of Ascorbic Acid in the Regulation of Root System Architecture

Regulation of the cellular redox state involves several redox metabolites. Among them, ascorbic acid (ASC) has a prominent role as a principal ROS scavenger. However, there is a growing body of evidence that the role of this substance in plants extends beyond its intensively explored antioxidant function.¹³ It has been demonstrated that high ASC levels are required for normal progression of the cell cycle in meristematic tissues where ASC was identified as a factor necessary for the G₁-S transition.^14–17 Besides its effect on cell proliferation, ASC stimulated cell elongation by increasing cell wall extensibility.^18,19

Because ASC is directly involved in the regulation of two processes that mediate morphogenic responses of root systems to nutrient availability, i.e., cell division and elongation, we addressed the role of this compound in the regulation of root system architecture by P availability. We found that P-starved plants accumulated significantly lower levels of ASC than those grown under high P levels. Moreover, supplementation of the growing medium with optimized concentrations of exogenous ASC partly reversed the inhibition of primary root elongation by low P concentration. On the other hand, the seedlings of vtc 1 mutant, containing reduced levels of ASC,²⁰ formed shorter roots when compared to the wild-type plants (Tyburski J, Tretyn A, unpublished data).

Analysis of mitotic activity in the primary root tips revealed that the effects of ASC level on primary root elongation may be, at least, partly due to the promotion of cell divisions in the primary root meristem. Exogenous ASC stimulated cell divisions in the tips of primary roots of seedlings grown at low P concentration. On the other hand, mitotic activity in the root tips of vtc 1 mutant seedlings was significantly reduced when compared to the wild-type plants (Tyburski J, Tretyn A, unpublished data).

In conclusion, our study has revealed that both, reactive oxygen species and antioxidants may possibly be involved in the developmental adaptation of the root system to low P availability.

Addendum to: Tyburski J, Dunajska K, Tretyn A. Reactive oxygen species localization in roots of Arabidopsis thaliana seedlings under phosphate deficiency. Plant Growth Regul. 2009;59:27–36. doi: 10.1007/s10725-009-9385-9.