Research Group Ragni
Secondary growth: the Arabidopsis hypocotyl as a model
General introduction to secondary growth:
Plant growth is characterized by two distinct developmental growth phases. Primary growth contributes to plant elongation and initial radial organization. It is driven by the apical meristems: the shoot apical meristem produces the areal part of the plant and the root meristem forms the root apparatus. Once the main body plan has been established, the plant undergoes secondary growth. Secondary growth results in the thickening of plant organ as the root and the stem. It does not only confer support to mechanical stress but it continuously produces new vasculature tissues. During secondary growth vasculature elements are formed by the cambium a secondary meristem (a meristem that is established post-embryonicaly). More in details the cambial stem cells proliferate and produce daughter cells that inward will differentiate into xylem (the so-called wood) and outward into phloem (Elo et al., 2009). Notably another meristem is produced during secondary development: the cork cambium/phellogen. The cork cambium is part of the periderm system (see figure1).
The emergence of secondary growth had a major impact on land colonization and shaped the extensive forest ecosystem that changed earth biosphere in the land we know. It facilitated the development of plants with increased height, improved photosynthetic output and it promoted the adaptation to different environmental habitats.
Why working on secondary development? Because in perennial dicotyledons xylem the so-called wood represents the principal form of biomass accumulation. Thus to promote a sustainable development is important to study the mechanisms underlying biomass accumulation.
Arabidopsis hypocotyl as a model
Despite being herbaceous Arabidopsis has been recently shown to be a good model to study secondary development and wood production (Chaffey et al., 2002; Ragni and Hardtke, in press; Sibout et al., 2008) (see figure 1). Recent studies have shown that the main regulatory machinery is conserved between herbaceous and woody plants. In Arabidopsis secondary growth can occur in roots, hypocotyls and stems. These organs do not display the same architecture and they all are established models to study secondary growth. The root and the hypocotyl share the same structural arrangement of the secondary tissues. This is very similar to Populus ssp.: from the procambium the cambium is formed as a continuous ring that produces inward xylem and outward phloem. The main difference between the root and the hypocotyl is that the root represents a developmental series (with newly initiated part at the tip and the thickest/mature part closer to the hypocotyl) whereas the hypocotyl presents a continuous structure as the hypocotyl stops to elongate after few days and then growth only radially. A key advantage of using the hypocotyl as a model is that elongation and radial growth are uncoupled. Furthermore it’s the organ that displays more secondary growth in Arabidopsis (Ragni and Hardtke, 2014).
Figure 1 Sketch of an Arabidopsis plant and cross-section of the hypocotyl.
In the literature in most studies secondary growth is measured as diameter length. Diameter length is a gross measure of secondary growth and it does not catch the complexity of the process at morphological level. Moreover it can even be even misleading i.e Col and Ler two Arabidopsis strains differ in hypocotyl diameter with Col being the bigger but it’s Ler that showed more xylem accumulation (Ragni et al., 2011). To overcome this problem we recently developed a new approach, to study hypocotyl development in a quantitative way, that we called the quantitative histology approach. It is base on: 1) a comprehensive anatomical atlas 2) High-ultra resolution images for extracting information 3) Automate pipeline to process and segment images 4) Automate cell type recognition using machine learning (Sankar et al., 2014) (see figure 2).
Fig 2 A An Example of ultra-resolution image. This image is composed by 12x12 images. B Example of segmented image and overlay of the original image with the segmented one.
1) Periderm development: biology and molecular mechanisms
The periderm is a three tissues system as the vasculature cambium composed by the cork cambium or phellogen, the cork or phellem and the phelloderm. The periderm starts with the formation of the cork cambium that will produce inward the phelloderm and outward the phellem/cork. In most species the phellloderm is composed by one to three layers of living cells (there are some exceptions in which no phelloderm is produced) whereas the number of layers of the suberized death cell of the phellem varies.(Pereira, 2011) In the majority of the stem of woody plants the cork cambium arises from an outer layer of the cortex, even thought in some species it originates from the epidermis or, alternatively, fairly deeply inside the stem, such as in the primary phloem. In the root and in the hypocotyl, it arises from the pericycle (see figure 3). In most trees, one periderm is functional only for a limited period and it’s replaced by a new functional periderm to the inner side. This mechanism leads to the accumulation of several dead tissues layer outside the functioning periderm forming the so call rhytidome or outer bark (Pereira, 2011).
Our main goals are the characterization of periderm development and the identification of the molecular players responsible for its establishment and maintenance using the Arabidopsis hypocotyl as a model. We aim to do: 1) An Anatomical ATLAS of Arabidopsis hypocotyl periderm 2) The transcriptome of the Arabidopsis periderm. 3) Find transcription factor that regulates periderm maintenance and establishment.
Fig 3 Periderm in the Arabidopsis hypocotyl.
2) Identification GA-downstream factors that play a role in xylem expansion
During Arabidopsis hypocotyl development we can distinguish two phases of secondary growth, an early phase in which xylem and phloem are produced at the same rate by the cambium and a later phase of xylem expansion, in which xylem is produced at higher rate, and fibers differentiate reminiscent of tree stems. Previously, it has been shown that flowering triggers the shift between the two phases (Sibout et al., 2008) (see figure 4A). Furthermore, grafting experiments suggested that a shoot-derived signal is necessary to trigger this xylem expansion. By contrast, in Arabidopsis neither flower formation nor elongation of the main inflorescence is required. Xylem expansion also does not depend on any particular flowering time pathway or absolute age (Sibout et al., 2008). Recently we have found that the gibberellin (GA), which has been shown to regulate cambial activity and wood deposition in trees, is limiting xylogenesis and that GA signaling is required locally to promote xylem expansion. In addition, the effect of GA was graft-transmittable suggesting that GA is the signaling molecule itself (Ragni et al., 2011) (see figure 4B).
We are currently investigating the downstream factors of DELLA signaling involved in xylem expansion.
Fig 4 A Secondary development in Arabidopsis hypocotyl. B Gibberellin promotes locally xylem expansion.
References quoted in this page
Chaffey, N., E. Cholewa, S. Regan, and B. Sundberg. 2002. Secondary xylem development in Arabidopsis: a model for wood formation. Physiologia Plantarum. 114:594-600.
Elo, A., J. Immanen, K. Nieminen, and Y. Helariutta. 2009. Stem cell function during plant vascular development. Seminars in Cell & Developmental Biology. 20:1097-1106.
Pereira, H. 2011. Cork: Biology, Production and Uses: Biology, Production and Uses. Elsevier Science.
Ragni, L., and C.S. Hardtke. in press. "Small but thick enough - the Arabidopsis hypocotyl as a model to study secondary growth". 2013 Physiologia Plantarum.
Ragni, L., K. Nieminen, D. Pacheco-Villalobos, R. Sibout, C. Schwechheimer, and C.S. Hardtke. 2011. Mobile Gibberellin Directly Stimulates Arabidopsis Hypocotyl Xylem Expansion. The Plant Cell Online. 23:1322-1336.
Sibout, R., S. Plantegenet, and C.S. Hardtke. 2008. Flowering as a Condition for Xylem Expansion in Arabidopsis Hypocotyl and Root. Current biology : CB. 18:458-463.
Dr. Laura Ragni
Center for Plant Molecular Biology - ZMBP
University of Tübingen
Auf der Morgenstelle 32
D - 72076 Tübingen, Germany
Phone: +49 (0)7071 - 29 76677
Fax: +49 (0)7071 - 29 5797