Inflorescence stems of Arabidopsis thaliana bend away from neighbours through a response controlled by phytochrome B
Plants have the ability to detect and respond to light of different wavelengths through a complex network of photoperceptive systems (Galvao & Fankhauser, 2015).
While blue and red light is highly absorbed by leaves, far red is reflected and transmitted through green tissues. Therefore, the section of the light spectrum corresponding to red and far red light provide important information to plants, especially about the presence or absence of neighbours, since the perception of light conditions characterized by a low red:far red ratio (R:FR) indicates the proximity of a dense shaded area, as opposed to an open landscape, which is characterized by high R:FR light conditions (Taylorson & Borthwick, 1969; Holmes & Smith, 1977).
The detection of the variation in R:FR is important particularly in shade-intolerant plants. Therefore, a series of developmental responses is triggered by the plants as soon as they perceive the presence of neighbours anticipating the shading and competing for light, which leads to an improvement of photosynthetical efficiency. This series of responses has received the name of Shade Avoidance Syndrome (SAS) and includes processes as petiole elongation (Hisamatsu et al., 2005; Casal, 2012) and enhanced growth of stems and internodes (Ballare et al., 1991; Devlin et al., 1998), both responses that typically contribute to escape from neighbours shade in terms of height. Few cases of lateral escaping (or negative phototropism) have been reported in response to far red light. Such are the cases of cucumber seedlings, which bend against a far red-reflecting canopy (Ballare et al., 1992) and leaves of some maize cultivars, which also escape from the low R:FR signal of surrounding plants (Maddonni et al., 2002). In addition, spatial distribution of A. thaliana rosette leaves was also found to be affected by the presence of neighbours (Crepy & Casal, 2014).
Most of the studies about these growth responses leading plants to escape away (or in height) from neighbours are focused in vegetative organs. Conversely, shade avoidance responses in flowers or inflorescences are usually understood only in temporal or developmental terms but not in spatial terms. For example, low R:FR ratio light conditions promote early flowering in A. thaliana (Halliday et al., 1994; Pigliucci & Schmitt, 1999), a strategy typically associated with stress avoidance (Stanton et al., 2000). The signal of neighbouring plants also affects floral display in A. thaliana by reducing lateral buds' development (Holalu & Finlayson, 2017). In addition, the development of taller inflorescence stems was described by Finlayson et al. (2010). Although it is possible that such a response might have a positive effect in pollination of plants growing in dense populated patches, it also has been reported that the promotion of stem growth by low R:FR conditions reduces grain yield in crops (Liebenson et al., 2002). Therefore it is suggested that there is a trade-off between resources allocated to the promotion of stem growth and those destined to yields, especially in densely-populated agricultural scenarios (Kebrom & Brutnell, 2007).
Plant responses to the variations in light ambient are controlled by a photoperceptive system, consisting on a complex network of photoreceptors. In the case of the model plant Arabidopsis thaliana (L.), five families of photoreceptors are known to date: UV-B radiation (315 nm) is perceived by the protein UVR8, UV-A and Blue light (320-500 nm) is signalized by three families of proteins: the phototropin family (PHOT1 and PHOT2), the crypto chrome family (CRY1 and CRY2) and the zeitlupe family (ZTL, FKF1 and LKP2). Finally, the phytochrome family, comprising five members (PHYA to PHYE) mediates responses to red and far red light (660 nm and 740 nm, respectively). The responses to the shade of surrounding vegetation are mainly controlled by the phytochrome family of photoreceptors. Phytochromes alternate into an active (Pfr) and an inactive (Pr) state when exposed to red or far red light respectively. Upon red light exposure, phytochrome B in its Pr form undergo a reaction of photoconversion to its Pfr form and translocate into the nucleus (Whitelam et al., 1998; Staiger, 2008), initiating a signaling pathway that ends in hypocotyl, petiole and stem growth inhibition (Ruberti et al., 2012). Conversely, after far red irradiation, the inverse reaction takes place, meaning that the Pfr form of phyB changes to Pr and emigrates from the nucleus to the cytosol; as a consequence, growth takes place. Mutant plants lacking one or more functional phytochromes show constitutive shade avoidance responses, e.g. they grow taller and have elongated petioles even in open landscapes (Whitelam et al., 1998).
In this article, the escape from a unidirectional signal of natural and simulated canopies is evaluated in inflorescence stems of A. thaliana. We hypothesized that inflorescences exposed to the unilateral signal of neighbours evoke an escape in height as well as a directional escape, which are controlled by phyB. The results we expect to find given that this hypothesis is correct are that plants grown beside natural and simulated canopies, compared to isolated plants, will exhibit: i) taller stems, ii) a bending response of the stem away from neighbours or iii) an asymmetric distribution of lateral branches (i.e. developed predominantly on the open side of the stem). In addition, if these effects are controlled by phyB we predict that these responses will be absent in phyB mutants lacking functional phyB protein.
Materials and Methods
In order to assess the above mentioned hypotheses we conducted two types of experiments, by which we attempted to determine; i) the effect of a natural canopy on inflorescence architecture and orientation ("natural canopy experiment", see details below), and ii) if a light ambient enriched in the far red portion of the spectrum is the main stimulus that promotes the responses of plants to their neighbours ("artificial far red light experiment", see details below). In both cases, the behaviour of mutant plants lacking functional phytochrome B (phyB) was compared with wild type (WT) plants in order to evaluate the role of phytochrome B in the responses.
For both kinds of experiments the growth conditions of the plant material were as follows. Seeds of A. thaliana plants were sown on 0.8% agar, stratified for 3-5 days at 4[degrees]C in darkness and then exposed to white light to induce germination. Then, seedlings were transferred to 180 [cm.sup.3] pots filled with two parts of perlite (Bio-Organic S.R.L, Mendoza, Argentine), two parts of peat moss Sphagnum (Bio-Organic S.R.L, Mendoza, Argentine) and one part of sand (Casa Forconi, Mendoza, Argentine), and watered each day with a solution containing 0.7 g/L of Hakaphos Red (COMPO, Spain).
Plants grew until flowering stage inside growth chambers under 12 hours light/12 hours dark at 200 [micro]mol x [m.sup.-2] x [s.sup.-1] of white light at 25[degrees]C. Once the inflorescence primordia were observable, the plants were transferred to a greenhouse and placed inside ventilated transparent closures of 1x3x1.5 meters covered by a UV-blue light-excluding filter (LEE Filters No. 767-Oklahoma Yellow) to avoid a potential phototropic effect of blue and ultraviolet light. Ventilation was achieved by leaving the lower part of the closures...