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Arabidopsis thaliana (L.) Heynh. (Brassicaceae) is a small annual distributed in open and disturbed habitats of temperate Eurasia (O’Kane & Al-Shehbaz, 1997). It has generally been accepted that the major part of its aboveground inflorescence stem does not show secondary growth under normal growth conditions. However, treatments that stop or delay flower development, such as manual clipping of flowers (Lev-Yadun, 1994) and changing of the photoperiod from long to short days (Chaffey et al., 2002), stimulate and prolong to a limited extent cambial activity in roots, hypocotyls, rosette-level short stems and/or the basal part of inflorescence stems in the Columbia (Col) wild-type accession. Likewise, other perturbations such as applying weights to the inflorescence stem (Ko et al., 2004) or intra-tissue tensions (Sehr et al., 2010) have a similar stimulating effect on the cambium. There is also extensive literature on plant hormones functioning as an essential stimulus in cambium initiation and activity, especially auxin (Aloni, 1987; Little et al., 2002; Agusti et al., 2011) and cytokinin (Matsumoto-Kitano et al., 2008; Hejátko et al., 2009). In addition, other hormones such as ethylene (Love et al., 2009), gibberellins (Björklund et al., 2007) and jasmonate (Sehr et al., 2010) can also stimulate secondary growth. Consequently, modulated expression of wood-forming genes still present in A. thaliana can cause a significant increase in wood formation (Oh et al., 2003; Ko et al., 2004; Groover, 2005). Interestingly, at least some key genes associated with the shoot apical meristem are also expressed in the cambial zone during secondary growth (Schrader et al., 2004), which might explain why there is a strong evolutionary pressure for these wood-inducing genes to be maintained in herbaceous plants (Spicer & Groover, 2010).
Compared with trees and even normal-sized shrubs, the amount of secondary growth in the inflorescence stems of the Col accession remains limited to only a few cell layers, and this hampers our understanding of the regulatory process involved in wood formation when A. thaliana is used as a model plant (Li et al., 2006). Consequently, the search for woodier A. thaliana mutants, such as cov1 (continuous vascular ring) (Parker et al., 2003) and hca (high cambial activity) (Pineau et al., 2005), and woodier transgenic plants over-expressing AtHB8 (Arabidopsis thaliana homeobox) (Baima et al., 2001) has had the aim of unraveling the genetic mechanism for wood formation. We recently described a double mutant (in the Col background) showing wood development throughout all stems and to a much larger extent than any A. thaliana mutant described to date (Melzer et al., 2008). In this double mutant, two MADS box genes, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and FRUITFULL (FUL), which both play a role in flowering time control, were knocked out. SOC1 integrates signals derived from photoperiod (Abe et al., 2005; Wigge et al., 2005), temperature (Searle et al., 2006), gibberellins and age-related signals (Wang et al., 2009; Lee & Lee, 2010), while FUL shows only a mild flowering time effect and plays additional roles in floral meristem identity, and carpel and leaf development (Ferrandiz et al., 2000; Teper-Bamnolker & Samach, 2005). In soc1 ful double mutants, flowering time is synergistically delayed compared with single mutants, indicating that both genes have redundant roles in flowering time control. Besides the shrub-like habit, the double mutants show a combination of perennial-like features, such as a prolonged plant lifetime of up to 18 months, the co-occurrence of active vegetative and reproductive meristems, and recurrent flowering cycles (Melzer et al., 2008).
Woody soc1 ful double mutants have been created in a Col background, implying that the double mutant is derived from an herbaceous Col accession and must therefore be called secondarily woody. There is ample molecular phylogenetic evidence for a herbaceous ancestry of secondarily woody taxa in many flowering plant families occurring on islands (therefore often referred to as insular woodiness; for example, Böhle et al., 1996; Lee et al., 2005) or island-like regions such as tropical mountain peaks (Jordon-Thaden et al., 2010). Furthermore, there are also wood anatomical characters – so-called paedomorphic features – that can be used to identify secondary or insular woodiness (Carlquist, 1962, 2009; Lens et al., 2009; Dulin & Kirchoff, 2010). Paedomorphic or juvenile wood features resemble characters of the primary xylem that are protracted into the more mature secondary xylem (= wood) of secondarily woody species. Likewise, delayed periderm formation is sometimes considered a paedomorphic bark feature (e.g. Cumbie, 1983), although cortical photosynthesis in primarily woody trees can also lead to delayed periderm formation.
The objectives of this study were: to describe the stem anatomy of the woody A. thaliana mutant (Supporting Information Notes S1); to look for characters in the woody mutant stem that point to a herbaceous descent; and to compare the wood anatomy of A. thaliana with that of other members of Brassicaceae that develop into woody shrubs in their natural environment (Carlquist, 1971; Kowal & Cutler, 1974; Schweingruber, 2006). A strong wood anatomical similarity between the soc1 ful double mutant and other woody Brassicaceae would provide evidence that the induced wood formation in the mutant is triggered by the ‘normal’ genetic pathway, and this would reinforce the idea that A. thaliana is a valid model organism with which to study the genetic mechanism behind wood formation in insular eudicots.
The boundary between herbaceousness and woodiness is vague
We found that basal inflorescence stems of Col plants expressed a limited amount of cambial activity in the intrafascicular regions (Fig. 1a,c,e,g), and started to develop an incomplete cambium ring in the interfascicular regions when inflorescence stems were between 15 and 20 cm in length (Fig. 1e). This limited secondary growth has been described before, although intraspecific variation exists: some studies have shown a whole ring of vascular cambium producing a few cell layers of wood in the basal stem parts, and cambial activity may reach up to 7 mm above the basal rosette in 30-cm-long inflorescence stems of the Col accession (Sehr et al., 2010). Nonetheless, this increased cambial activity remains limited compared with the woodier soc1 ful mutant (Fig. 1f,h) and can be linked to the more juvenile stages (Fig. 1b,d). The presence of secondary tissues in the Col accession agrees with numerous anatomical studies on herbaceous plants showing a continuous range of limited wood formation in basal stem parts (cf. Dulin & Kirchoff, 2010; references cited therein). Therefore, the boundary between the terms ‘herbaceousness’ and ‘woodiness’ is fuzzy from an anatomical point of view.
While the Col accession showed negligible wood development in the inflorescence stems, the double mutant can be considered a truly woody shrub (Melzer et al., 2008). However, roots of the double mutant did not show an increase in secondary growth to the same extent as in stems, suggesting that other key regulatory genes are involved in wood formation within roots. The increasing difference in wood development with age between the aboveground and underground tissues of the double mutants does probably inhibit the growth of these plants for several years.
Striking wood anatomical similarity between A. thaliana and other members of Brassicaceae
The microscopic wood structure of the soc1 ful double mutant was found to be remarkably similar to that of the other (secondarily) woody Brassicaceae investigated (Figs 2, S1 and Notes S1), showing notably short vessel elements (usually < 200 μm), exclusively simple vessel perforations (Figs 2c, S1b), solitary vessels in combination with vessels arranged in (often radial) multiples, vestured intervessel pitting (Figs 2d, S1d), axial parenchyma adjacent to vessels, and fibres with simple pits or with reduced pit borders (Carlquist, 1971; Kowal & Cutler, 1974; Schweingruber, 2006). Noteworthy was the presence of vestured pits in the double mutant and other woody Brassicaceae genera. Within the order Brassicales, vestures are present in only a few families, including Brassicaceae and its two most closely related families, Capparaceae and Cleomaceae, which re-emphasizes the informative phylogenetic value of vestured pits at high taxonomic levels (Jansen et al., 2001, 2004). One of the minor anatomical differences in wood structure between soc1 ful mutants and other woody Brassicaceae was the absence of rays (Fig. 2a) vs the presence of multiseriate rays, including predominantly square to erect ray cells (Fig. S1a,c). This distinction is only a minor one, however, and has been observed in many secondarily woody taxa (Carlquist, 1962, 2009; Lens et al., 2007, 2009). Furthermore, it is known that rayless species with relatively thin stems, such as A. thaliana, may develop rays at a later stage when the stem reaches larger diameters (Carlquist, 1970).
The fact that the wood anatomical characters of the A. thaliana double mutant were nearly identical to the anatomy of other woody Brassicaceae strengthens our assumption that inactivation of both the SOC1 and FUL genes triggers the ‘normal’ pathway leading to increased wood formation. The observed anatomical similarity supports the hypothesis that the genetic mechanisms underlying the developmental processes of wood development may potentially be highly conserved between woody and herbaceous plants (Kirst et al., 2003; Schrader et al., 2004; Groover, 2005; Spicer & Groover, 2010), implying that knock-out mutants would be perfect models for secondary growth and wood developmental research (Chaffey et al., 2002; Nieminen et al., 2004).
Paedomorphic wood features in the soc1 ful mutant and other woody Brassicaceae indicate herbaceous descent
A second strong anatomical argument for the activation of the ‘normal’ genetic pathway leading to extensive wood formation in the double mutant is the expected occurrence of two paedomorphic wood features, that is, the absence of rays (Figs 1h, 2a) and the continuously decreasing length-on-age curve of vessel elements (Fig. 2e); the delayed periderm formation in the double mutant (Fig. 2a) might be considered a third paedomorphic bark feature, and is also present in some woody Brassicaceae (Fig. S1a). According to Carlquist (1962, 2009), the most convincing wood character that identifies secondarily woody species is the flat or continuously decreasing length-on-age-curve of vessel elements: after a strong decrease in vessel element length in the primary xylem and first formed secondary xylem from 450 to 150 μm, the length stabilizes and shows an extremely low value of c. 100 μm (Fig. 2e). A similar situation was observed in the three other woody Brassicaceae species studied (Fig. S1e). Although we are aware that the maximal stem diameter in our double mutant might not be large enough (5 mm) to reach full maturity in its wood structure, we consider Fig. 2(e) a representative length-on-age curve. This is based on Bailey (1920), who demonstrated that the range of vessel elements remains almost constant in the secondary xylem in species that are characterized by so-called derived vessel elements, that is, very short vessel elements (< 300 μm on average) combined with exclusively simple perforations (cf. Carlquist, 1962). A hydraulic explanation of the variation in vessel element length during secondary growth has not been forthcoming: it is assumed that the length of entire vessels is more important in the water transport mechanism of plants, and greatly overwhelms the importance of vessel element length (e.g. Lens et al., 2011).
The soc1 ful double mutant of A. thaliana produced substantial secondary growth throughout all aboveground stems, whereas in the Col accession only a few cell layers of wood were produced at the base of old stems. This increased wood formation may be linked to inactivation of the flowering time genes SOC1 and FUL (Melzer et al., 2008). These two genes could play a crucial regulatory role in triggering genetic mechanisms that lead to insular woodiness outside A. thaliana, and this ‘simple’ two-gene loss could explain why many nonrelated herbaceous families have independently evolved into woody insular lineages throughout the world. However, SOC1 and FUL might not be the only suppressors of cambium formation; other upstream, downstream or parallel-acting (positive or negative) regulators could be even more important. Therefore, it is evident that many missing pieces of this intriguing genetic puzzle leading to insular woodiness in eudicots need to be found before we can better understand one of the most fascinating developmental aspects of plants (cf. Lev-Yadun, 1994; Chaffey et al., 2002; Nieminen et al., 2004).
S.M. was a senior fellow (SF/10/007) of the Research Fund of the K.U. Leuven.