Stem transcriptome during shade avoidance

Whilst the most conspicuous response to low red to far-red ratios (R:FR) of shade light perceived by phytochrome is the promotion of stem growth, additional, less obvious effects may be discovered by studying changes in the stem transcriptome. Here we report rapid and reversible stem transcriptome responses to R:FR in tomato ( Solanum lycopersicon ). As expected, low R:FR promoted the expression of growth-related genes, including those involved in the metabolism of cell-wall carbohydrates and in auxin responses. In addition, genes involved in flavonoid synthesis, isoprenoid metabolism and photosynthesis (dark reactions) were overrepresented in clusters showing reduced expression in the stem of low R:FR-treated plants. Consistent with these responses, low R:FR decreased the levels of flavonoids (anthocyanin, quercitin, kaempferol) and selected isoprenoid derivates (chlorophyll, carotenoids) in the stem, and severely reduced the photosynthetic capacity of this organ. However, lignin contents were unaffected. Low R:FR reduced the stem levels of jasmonate, which is a known inducer of flavonoid synthesis. The rate of stem respiration was also reduced in low R:FR-treated plants, indicating that by downsizing the stem photosynthetic apparatus and the levels of photoprotective pigments under low R:FR, tomato plants reduce the energetic cost of shade-avoidance responses.

Due to the optical properties of green leaves, canopy shade is characterized by low red / far-red ratios (R:FR) of the light. One of the most conspicuous responses of plants exposed to low, compared to high R:FR, is the promotion of stem growth (Smith, 1982;Casal and Smith, 1989;Ballaré, 1999;Morelli and Ruberti, 2002;Franklin and Whitelam, 2005;Casal, 2012). A taller stem exposes the leaves to higher and better light strata within the canopy. Conversely, leaf growth may increase, decrease or remain unchanged when the plants receive low R:FR.
In sparse canopies, the horizontal leaves are exposed to the high R:FR of full sunlight while the vertical stems can receive low R:FR, caused by selective reflection of FR by the green foliage. This provides an early warning signal of the presence of neighbours (Ballaré et al., 1987). At later stages of canopy development the leaves become mutually shaded and exposed to low R:FR. The low R:FR reaching only the stem causes a promotion of stem growth that is rapidly reversed to the pre-stimulation values upon return to high R:FR. However, when the low R:FR also reaches the leaves, the promotion is more persistent and only returns to the pre-stimulation values 24 h after the plants are exposed to high R:FR (Casal and Smith, 1988). Therefore, there is a positive correlation between the threat indicated by the shade signals and the persistence of the growth response.
In Arabidopsis seedlings grown under continuous white light for 7 d (at which point they were producing their first true leaves), transfer to low R:FR significantly promotes the expression of cell wall-cell elongation-, cell division-and auxinassociated genes (Devlin et al., 2003). In Arabidopsis plants grown for 19 d under continuous white light before transfer to darkness, a pulse of far-red light given at the end of white light promoted the expression of auxin-and brassinosteroid-responsive genes both in the leaf petioles and blades, despite the contrasting growth responses of these parts of the leaf to far-red light (Kozuka et al., 2010). Conversely, several xyloglucan endotransglucosylase-hydrolase (XTH) genes showed increased expression in the petiole but not in the blade, which correlates with the growth response (Kozuka et al., 2010).
Previous studies have described organ-specific transcriptome responses observed when dark-grown seedlings of Arabidopsis (Ma et al., 2005;López-Juez et al., 2008) or soybean (Li et al., 2011) are exposed to light for the first time. Despite the large stem-growth response to R:FR, our knowledge of stem transcriptome responses to R:FR is very limited. Here we used light-grown tomato (Solanum lycopersicon) seedlings to investigate the stem transcriptome responses to R:FR. By comparing the stem (first internode) and the first pair of true leaves we identified shared and organ-specific responses. We relate the differential stem and leaf transcriptome responses to organ-selective hormonal and physiological responses.

Shared and specific stem and leaf transcriptome responses to low R:FR
Plants of tomato were grown for 24 d under continuous white light. When the first internode (the stem between the cotyledonary node and the first leaf pair node) was 6.4 ±0.4 mm (mean, SE), half of the plants were exposed to supplementary FR reaching the leaves and the stem from both sides, to lower the R:FR of the horizontally propagating light from 4.6 (white light alone) to 0.05 (white light plus FR). Four days later (see photograph of representative plants in Fig. 1A), the first internode and the first pair of leaves were harvested separately. Total RNA was extracted from these samples, processed and hybridized to tomato Affymetrix microarrays. Microarray data were filtered by presence criteria and subjected to factorial ANOVA with R:FR (high or low) and organ (stem or leaves) as main factors. The genes showing significant effects of treatments (p <0.05, q <0.003) were divided in three groups according to the significance of the effects of R:FR and its interaction with organ (Table S1) Table S1). Most genes of this first group also showed significant effects of organ, which were additive to the R:FR effects. A second group included genes showing differential responses to R:FR in the stem and leaves (significant interaction, p <0.05, q <0.09). Two clusters of this gene group showed promotion of expression by low R:FR in the stem (I1, I5), three clusters showed promotion of expression by low R:FR in the leaves (I2, I8, I11), two clusters showed inhibition of expression by low R:FR in the stem (I3, I9) and three clusters showed inhibition of expression by low R:FR in the leaves (I6, I7, I10) (Fig. 1B, Table S1). Few genes showed opposite responses (quantitatively modest) in stem and leaves (cluster I4). Some genes showed reduced expression in response to R:FR in both organs but the effects were significantly higher in the stem (cluster I9). A third, residual group of genes showed no interaction, no significant effects of R:FR and significant effects of organ, and were not further analysed (Table S1).
The total number of genes showing statistically significant responses to low R:FR in the same direction in both organs was larger than the number of genes responding significantly more in the leaves or the stem (Fig. 1D). However, the latter picture was inverted when the analysis was restricted to those genes with statistically significant responses with a magnitude of at least two fold. In other words, organindependent changes are more numerous but the largest changes in gene expression are organ specific (Fig. 1D).

Rapid and reversible stem transcriptome responses to low R:FR
A second microarray experiment was designed to investigate the early kinetics of stem transcriptome responses to low R:FR. Plants were grown under continuous white light, exposed to supplementary FR for either 1 or 4 h and harvested 0, 1 or 3 h after returning to high R:FR (seven conditions, including the control never exposed to low R:FR). To minimise the impact of circadian rhythms the plants were grown under continuous white light and harvested simultaneously. Microarray data were filtered by presence criteria and subjected to ANOVA (Table S2). The genes showing significant effects of treatments (p <0.01, q < 0.10) were used for cluster analysis. A total of 424 genes (clusters S1-S5) showed increased and 410 genes (clusters S6-S9) showed reduced expression in response to low R:FR (Fig. 1C, Table S2). These clusters differed in the kinetics of response, from those that responded rapidly to the transfer from high to low R:FR and showed a rapid reversal of the response after transfer to high R:FR (clusters S2, S8 and S6, the latter with transient overcompensation after the return to high R:FR) to those showing a gradual response to low R:FR and to subsequent high R:FR (clusters S7 and S9) (Fig. 1C). Many genes were significantly affected by both 4 h and by 4 d of low R:FR compared to high R:FR controls. These genes showed a strong correlation in the extent of relative response at both time points ( Fig. 1E), supporting the robustness of the reported changes in gene expression.

Overrepresented GO terms
We searched for overrepresented GO terms within each cluster (Fig. 1). The overview of both experiments revealed three major areas of transcriptome response in the stem: a) Cell-wall carbohydrate process related genes are overrepresented in Cluster I5, b) Flavonoid process related genes are overrepresented in clusters I3, and S7, and c) Photosynthesis related genes and photosynthetic pigment related genes (e.g.
carotenoid metabolism genes within the GO terms "isoprenoid biosynthetic process" and "steroid metabolic process") are overrepresented in clusters S7, S8 and S9. In subsequent sections we investigate these functions in further detail. Transport-related genes were overrepresented in cluster I4 but the responses were small in magnitude.
The leaves showed photosynthetic-and translation-related genes overrepresented in cluster I6 and flavonol-related genes overrepresented in cluster I8 (Fig. 1). The clusters describing organ-specific responses contain most of the genes with a significant effect of R:FR of at least two-fold and yielded most of the overrepresented GO terms.

Low R:FR differentially affects stem and leaf growth
The growth rate of the stem showed a rapid promotion by low R:FR. When the plants were returned back to high R:FR, the rate of stem growth also returned rapidly to the pre-stimulation values ( Fig. 2A). However, following a short (1 h) exposure to low R:FR, the stem growth rate showed an overcorrection upon the return to high R:FR leading to Some observations require clarification before continuing with the main stream analysis of this work. Previous results had shown that mustard (Casal and Smith, 1988) and tomato (Casal et al., 1995) plants exposed to low R:FR for more than 3 h have elevated rates of stem growth after transfer to high R:FR. This was confirmed under the current conditions, as upon transfer to high R:FR, the stem of plants previously exposed to 4 h low R:FR grew more during the subsequent 20 h (1.7 ±0.2 mm, mean ±SE, P <0.01, n= 42) than the plants previously exposed to only 1 h low R:FR (1.1 ±0.1 mm) or high R:FR controls (1.0 ±0.1 mm). However, this long-term promotion was not observed immediately after the 4 h of low R:FR ( Fig. 2A). showed reduced levels in the stem of low R:FR-treated plants.

Low R:FR differentially affects stem and leaf flavonoids
The flavonoid synthesis genes that show reduced expression in the stem of tomato plants exposed to low R:FR did not respond to low R:FR in whole Arabidopsis seedlings (based on the analysis of publicity available data (Sessa et al., 2005)). In  (Sessa et al., 2005). In other words, this flavonol biosynthesis gene increases its expression in response to low R:FR in the leaves of tomato (Fig. 3) and in leaf-rich Arabidopsis samples.

Low R:FR differentially affects photosynthesis and respiration in the leaf and stem
Photosynthesis-related genes are overrepresented in clusters S7 and S9, showing a rapid reduction in stem expression in response to low R:FR (Fig. 1C). These genes are linked mainly to dark reactions (Calvin cycle) (Fig. 4D). In addition, genes related to isoprenoid biosynthesis and steroid metabolism, which represent steps upstream chlorophyll and carotenoid biosynthesis, are respectively overrepresented in clusters S8 and S9 (Fig.1C). These observations prompted us to measure the rates of net carbon dioxide exchange against irradiance in the stem and in the leaves of tomato plants exposed to low R:FR for 4 d. In the controls exposed to high R:FR, the stem showed a significant photosynthetic capacity, equivalent to approximately 1/3 of the leaf capacity at saturating irradiance ( The levels of JA were significantly reduced in the stem of low R:FR-treated plants (Fig. 5), showing a pattern consistent with that of anthocyanin levels and the expression of anthocyanin-synthesis genes. Noteworthy, the expression of the 12-Oxophytodienoate-10,11-Reductase gene involved in JA synthesis (Schaller et al., 1998;He et al., 2002) showed a rapid decrease in the stem of low R:FR-treated plants (Table S2). The levels of ABA increased in the leaves of low R:FR-treated plants, those of ACC showed a similar promotion by low R:FR in the stem and leaves and IAA contents were not significantly affected by R:FR (Fig. 5).

Light-dependent induction of anthocyanin by JA
To investigate the physiological role of the stem-specific reduction in JA content we selectively added JA to the stem of plants exposed to either high or low R:FR.
Exogenous JA increased anthocyanin levels in the high R:FR controls (Fig. 6A), suggesting that the higher levels of endogenous JA under this light condition (compared to low R:FR) would be a requisite for the observed high anthocyanin levels.
Exogenous JA had no effects in low R:FR-treated plants (Fig. 6B), suggesting that the promote stem growth at low R:FR (Fig. 6B). Therefore, JA also reduced shade avoidance in our system (note reduced difference between high and low R:FR in JAtreated stems).

DISCUSSION
When plants are shaded by neighbours, the photoassimilates that fuel growth become limited by light availability. In response to the low R:FR signals of dense vegetation the growth of the stem is promoted to place the leaves at a higher position, with better chances to capture light for photosynthesis (Smith, 1982;Casal and Smith, 1989;Ballaré, 1999;Morelli and Ruberti, 2002;Franklin and Whitelam, 2005;Casal, 2012 Genes involved in early steps of the flavonoid-synthesis pathway and downstream genes leading to anthocyanin synthesis showed comparatively high levels of expression in the stem of plants exposed to high R:FR, a strong (and often rapid) reduction in response to low R:FR, and lower expression levels in the leaves (Fig. 3).  (Tegelberg et al., 2004). Actually, in tomato leaves we observed increased quercetin and rutin levels in response to low R:FR, which correlated with the promotion of expression by low R:FR of some flavonol-related genes in these organs (Fig. 3). Low R:FR also reduced the expression of early genes in the isoprenoid and steroid pathways in the stem (Fig. 1C). Some of the downstream products of the latter genes, such as chlorophyll and carotenoids, were also severely reduced in the stem (Fig. 4E, F) while ABA, which is another downstream product, was unaffected in this organ ( selectively to the stem increased anthocyanin levels in the stem of seedlings exposed to high R:FR but not of those exposed to low R:FR (Fig. 6). These results suggest that anthocyanin levels are high under high R:FR due to increased JA levels and increased sensitivity to JA.
In Arabidopsis, the coi1, jar1, jin1, and jai mutants show enhanced promotion of hypocotyl elongation by low R:FR, indicating that JA biosynthesis and signalling genes reduce shade-avoidance responses (Robson et al., 2010). Application of JA to the stem of tomato yielded a result consistent with the latter observation as it reduced the response to low compared to high R:FR (Fig. 6A). Although in contrast to the case of Arabidopsis, this reduced response in tomato was caused by enhanced growth under high R:FR and not reduced growth under low R:FR. The observed reduction of JA levels in the stem of tomato exposed to low R:FR could therefore be part of a positive feed-back loop of shade-avoidance responses in this organ.
In tomato, non-leaf green organs, including the stem, are potentially quite active photosynthetically (Hetherington et al., 1998). However, in some cases the tomato stem shows no net carbon dioxide exchange because its photosynthetic capacity can be enough to re-fix the respired carbon dioxide (Xu et al., 1997). Here we show that in plants grown under high R:FR the stem certainly has significant photosynthetic capacity, equivalent to approximately one third that of the leaves. However, under low R:FR this capacity is substantially reduced and net rates of carbon dioxide exchange are close to zero at irradiances above the compensation point (Fig. 4B). There would be no benefit in maintaining the maximum potential of the photosynthetic apparatus when shade limits the expression of this potential. The reduction of photostnthesis has a non-stomatic origin (Fig. 4C) and could be caused by limitations in the Calvin Cycle ( Fig. 4D). In addition, the rates of mitochondrial respiration are also strongly reduced by low R:FR.
Taken together, the results indicate that in response to low R:FR the stem photosynthetic apparatus is downsized. In the stem, photosynthetic and photoprotective pigment abundances decline, the photosynthetic capacity becomes minimized and a positive relationship between R:FR and JA levels appear as an important component of the control of anthocyanin content. In turn, these changes reduce the energetic cost of producing and maintaining a longer stem and this is manifested in lower respiration rates. The low R:FR-induced savings are very selective as they mainly involve the stem and not the leaves and, within the stem, they involve processes linked to light (which is becoming scant) and not processes related to the stem physical support function, as lignin concentrations are not reduced.

Light treatments
Twenty four-day-old plants were separated in two groups. The control group continued to grow under the high R:FR (= 4.6) conditions described above. The low R:FR (= 0.05) group received continuous supplementary far-red light (7.8 µmol m -2 s -1 ) provided from both sides by incandescent lamps in combination with a water filter (10 cm width), a red filter (no. 026, Lee Filters), and three blue acrylic filter (2.5 mm thick, Paolini 2031, Buenos Aires).

Stem and Leaf growth.
The length of the first internodes (the stem between the cotyledonary node and the first true leaf node) was measured with a ruler to the nearest millimetre. Dry weight was determined after drying at 70 ºC during 1 d. Leaf area was measured with a LI-3100 meter ( LI-COR, Lincoln, Nebraska). For detailed kinetics, the plants were photographed with a Canon Power Shot A520 camera and the images of the different time points were aligned using Photoshop 7.0 to record internode length increments.

Microarray Experiments
In a first experiment, the first pair of leaves and the first internode of both low R:FR and control plants (three biological replicates) were harvested in liquid nitrogen, and total RNA was extracted with RNAEasy Plant Mini Kit (Qiagen). In a second experiment, only the first internode was harvested at different times after transfer from high to low and from low to high R:

Lignin
Relative lignin levels were determined using the thioglycollic acid method (Bruce and West, 1989). Samples containing 200 mg of plant material were stored at -20 ºC and subsequently homogenized. Absolute methanol (25 mL) was added to the samples, which were vacuum filtered and rinsed with further methanol on filter paper. The solid material was dried at 60 ºC for at least 24 h, combined with 5 ml 2 M HCl and 0.5 ml 80% (v/v) thioglycollic acid (Riedel-deHean, Germany) and placed in boiling water for 4 h. After centrifugation (30000 g, 10 min, 4 ºC) the pellets were washed with distilled water, re-suspended in 5 ml 0.5 M NaOH and incubated at 25 ºC for 18 h. After further centrifugation, 1 mL of concentrated HCl was added to the supernatants and incubated for 24 h at 4ºC. Finally, the samples were centrifuged (1000 g, 10 min, ca 25ºC) and the pellet re-suspended in 3 ml of 0.5 M NaOH, where absorbance was determined spectrophotometrically at 280 nm.

Flavonol levels
Samples were ground with liquid nitrogen and flavonoids extracted in methanol:hexane (1:0.5 v/v per 0.1 g fresh weight) and stirred overnight at 25 °C with rotation at 200 rpm, followed by centrifugation at 13,000 rpm for 10 min. The methanol fraction was dried in a rotary evaporator and the sample resuspended in 400 µL of methanol (protocol based on (Torres et al., 2005). Resuspended extracts (5μL)

Chlorophyll, carotenoid and Anthocyanin levels
The samples were harvested in 1ml Acetone (pure solvent) and incubated in darkness at -20 ºC for at least 3 d. Absorbance was measured at 661.6, 644.8, and 470 nm to calculate chlorophyll and carotenoids levels (Lichtenthaler and Buschmann, 2001). For anthocyanin levels, plant material was extracted in 1 ml 1% (w/v) HCl methanol.

Leaf Photosynthesis
A portable gas-exchange system (LI-COR 6400; LI-COR) was used to obtain curves of CO 2 exchange against PAR provided by the red and blue diode gas -exchange system (6400-02B LED light source; LI-COR) in fully expanded first leaves and first internode stems. The area included in the 6 cm 2 chamber was recorded for each sample.

Analysis of hormone abundances
Freeze dried stem and leaf tissue was finely chopped with a razor blade and the masses were measured (2.74 to 3.2 mg dry weight). A mixture of stable isotope Two ion pairs were monitored for each hormone, and the larger fragments were used for quantification 195,224,226;136,189,195;166,190,194;216,245, 249 m/z).

Supplemental Data
The following materials are available in the online version of this article.
Supplemental Table S1. Stem and leaf transcriptome responses to R:FR. early stem expression responses to low R:FR (S1-S9). In B and C, data are means and SE of all the genes represented in the cluster (the number of genes is indicated in brackets), clusters with less than 30 genes are not included (see Table S1) and