A systems biology approach to dissect the contribution of brassinosteroid and auxin hormones to vascular patterning in the shoot of Arabidopsis thaliana

Abstract

Systems biology can foster our understanding of hormonal regulation of plant vasculature. One such example is our recent study on the role of plant hormones brassinosteroids (BRs) and auxin in vascular patterning of Arabidopsis thaliana (Arabidopsis) shoots. By using a combined approach of mathematical modelling and molecular genetics, we have reported that auxin and BRs have complementary effects in the formation of the shoot vascular pattern. We proposed that auxin maxima, driven by auxin polar transport, position vascular bundles in the stem. BRs in turn modulate the number of vascular bundles, potentially by controlling cell division dynamics that enhance the number of provascular cells. Future interdisciplinary studies connecting vascular initiation at the shoot apex with the established vascular pattern in the basal part of the plant stem are now required to understand how and when the shoot vascular pattern emerges in the plant.

The plant vascular system is responsible for the long-distance transport of water, solutes and molecules throughout the plant, being essential for plant growth and development. It is formed by two different functional tissues: the xylem, which transports water from roots to aerial organs, and the phloem, through which nutrients and photosynthetic products and signaling molecules are transported.

During embryogenesis, the vasculature is characterized as an undifferentiated procambial tissue in the innermost part of the plant embryo.¹ Later in development, the procambium (i.e., a group of pluripotent stem cells²) begins to divide and differentiate into xylem and phloem tissues through oriented cell divisions. In the shoot, procambium generates xylem tissue centripetally and phloem tissue centrifugally, driving the formation of collateral vascular bundles around it.^3,4 In the inflorescence stem of the model plant Arabidopsis, the radial pattern of the vasculature exhibits a periodic organization made by the alternation of vascular bundles and interfascicular fibers, which altogether form the vascular ring (Fig. 1A).

Figure 1.

Vascular patterning in Arabidopsis shoot inflorescence stem. (A) Radial section of DR5::GUS expression at the base of the inflorescence stem in Arabidopsis Col-0 plants. (B) Computer simulation result for auxin concentration ([Auxin]) in arbitrary units (a.u.) along a ring of cells; x and y stand for spatial coordinates. Auxin is distributed in maxima which, according to the model hypothesis, position vascular bundles. (C) Longitudinal section of Arabidopsis Col-0 wild-type plant at the most apical zone, immediately below the shoot apical meristem. Arrows point to xylem strains coming from the lateral organs.

Previous studies have documented the importance of plant hormones such as auxin and BRs in vascular cell differentiation and patterning.⁵ Defective polar auxin transport distorts shoot vascular patterning^6,7 and BR loss-of-function mutants exhibit few vascular bundles.^8,9 But how do these hormones control shoot vascular patterning? In order to answer this question, we used both quantitative measurements of vascular phenotypes and computational modeling.¹⁰

Auxin Polar Transport, Auxin Maxima and Shoot Vascular Patterning

Auxin is an essential hormone for plant development and it plays important roles in vascular differentiation.^11–14 According to the auxin-flow canalization hypothesis,¹⁵ auxin flow drives continuous strands or paths where the vasculature emerges, by enhancing its own transport. In leaves, it has been shown that the expression of auxin response marker DR5 and of auxin efflux carrier protein PIN1 precedes vascular induction.¹⁴ Recent work has shown that PIN1 exhibits a dynamic expression pattern that involves its lateral polarization towards the future leaf midvein.¹⁶ Furthermore, it is the polar localization of auxin efflux carriers that directs auxin in these cells.^14,17 Indeed, auxin maxima driven by the polarity of efflux carriers are emerging as a common module for Arabidopsis patterning in leaves, shoots and roots.¹⁸ In the shoot apical meristem, auxin maxima induce the initiation of organ primordia, such as secondary meristems and leaves during the generation of phyllotactic patterns.^19–21

To address how auxin polar transport controls shoot vascular patterning, we formulated a mathematical model for auxin transport dynamics,¹⁰ which partially captured the complexity of previously proposed models.^22,23 A hypothesis of the model is that auxin is distributed in maxima, which in turn direct vascular bundle formation in shoots (Fig. 1B). To support this hypothesis we analysed the expression pattern of auxin-response element DR5::GUS²⁴ in shoot inflorescence stems. Our results supported the plausibility of the model hypothesis and showed that DR5::GUS expression is within procambial and xylem tissues, colocalizing with vascular bundles (Fig. 1A).

To evaluate the role of auxin polar transport we analyzed computationally the effect of reducing active efflux transport rates. Our results showed that auxin dynamics slows down leading to more homogeneous and distorted auxin distributions. In turn, our analysis of the shoot vascular phenotypes of pin1pin2 double mutants and of plants treated with auxin transport inhibitor NPA revealed disorganization of the shoot vascular pattern with increased vascular bundles in these plants, in agreement with the model predictions.

Another prediction raised by the computational model is that changes in overall auxin levels should not alter the distribution of auxin maxima and, thereby, nor the shoot vascular phenotype. In agreement, we found that the auxin overproducing mutant yucca²⁵ exhibits no differences in the number of vascular bundles nor in the number of cells across the vascular ring compared to the wild type.

Brassinosteroids, Vascular Bundle Number and Cell Divisions

BRs have been shown to play an important role in vascular cell differentiation in xylogenic cell cultures from Zinnia.^26,27 The initial evidences for a role of BRs in Arabidopsis vascular development come from the characterization of BR-deficient^8,28 and perception mutants,²⁹ which exhibit a reduced number of vascular bundles.^9,30 The identification of novel BRL (BRI1 RECEPTOR LIKE) receptors in Arabidopsis, which are predominantly expressed in the vascular tissues, revealed that BR signal transduction in vascular cells promotes xylem differentiation in the plant shoot.²⁹

To investigate the role of BRs on shoot vascular patterning, we carried out a comprehensive vascular analysis at the shoot inflorescence of mutants with reduced BR-signaling or synthesis.¹⁰ Our results confirmed that these mutants exhibit a reduced number of vascular bundles compared to wild-type plants. In contrast, we found that mutations increasing BR-signaling or levels lead to the formation of higher numbers of vascular bundles. Thus, we concluded that BRs control the shoot vascular pattern by modulating the number of vascular bundles.

To further assess how BRs may promote the formation of vascular bundles, we turned into our mathematical model. The model indicated two distinct ways of controlling vascular bundle number: by changing the total number of cells across the vascular ring and by changing the size (measured through number of cells and not distances) of vascular units (i.e. vascular bundle and clockwise contiguous interfascicular cells). The quantification of the number of procambial cells forming the vascular ring in BRs mutants pointed out a strong correlation between vascular bundle and cell numbers, while differences in size could not account per se for the changes in vascular bundle number. Therefore, we concluded that BRs enhance vascular bundle number by increasing the amount of provascular cells. Intriguingly, this result suggests a new role of BRs in modulating procambial cell divisions.

Shoot Apical Meristemand Vascular Patterning Initiation

Our actual study has taken the established vascular pattern at the base of the inflorescence stem of Arabidopsis to unravel how auxin and BRs control shoot vascular patterning. Yet, we still do not know where, when and how the shoot vascular pattern is primarily induced.

The shoot vascular pattern is continuously formed in apical regions of the shoot. The inspection of the vascular pattern along the stem, from apical to basal parts, can provide a spatiotemporal representation of shoot vascular patterning.³¹ In Col-0 wild-type plants the differentiating xylem cells can be observed at approximately ∼100 µm below the shoot apex.¹⁰ Longitudinal sections of the same plants show the connection between the stem vasculature and the vascular strands of secondary stems and floral organs (Fig. 1C). Recently, it has been proposed how PIN1 polarization and auxin flow dynamics can drive the connection between leaf midveins and the shoot vasculature.¹⁷ Yet, it is not known whether the differentiating shoot vascular tissue is directing vascular patterning in more apical parts or whether the emergence of lateral organs and their vasculature, controlled by the SAM, is dictating shoot vascular patterning. The similarities between the mechanisms proposed for the role of auxin transport in phyllotaxy^22,32 and shoot vascular patterning¹⁰ suggest to primarily analyse the latter scenario.

One of the limitations in the study of vascular patterning is the technical difficulty to observe the vascular tissues since they are the most inner layers in the whole plant. The use of histological sections has proven to be an excellent tool to observe shoot vascular phenotypes. However, in order to advance in our understanding of shoot vascular patterning, new and more advanced tools^33,34 that can depict the spatio-temporal conformation of the stem vasculature by early markers, such as ATHB8,³⁵ and PIN1,¹⁴ will be very useful. Cell-type specific analyses together with genetic manipulations, quantitative measurements and modeling will surely facilitate our comprehension of shoot vascular pattern initiation.

Addendum to: Ibañes M, Fàbregas N, Chory J, Caño-Delgado AI. Brassinosteroid signaling and auxin transport are required to establish the periodic pattern of Arabidopsis shoot vascular bundles. Proc Natl Acad Sci. 2009;106:13630–13635. doi: 10.1073/pnas.0906416106.