Root-to-Shoot Communication to Modulate Source-Sink Relationship in Tomato Depends on Phytochromes

Phytochromes have been reported as strategic photoreceptors that can modulate the partition of photoassimilates between source and sink. However, so far, it is unknown whether phytochrome accumulation in the root is part of the control mechanisms of the source-sink relationship that modulates root and shoot growth. Thus, the objective of this work was to investigate phytochrome involvement in the source-sink relationship and in the vegetative and reproductive development of tomato plants (Solanum lycopersicum L. cv. Micro-Tom or MT). The experimental design was completely randomized with four treatments, provided by grafting combinations between aurea (au), which is phytochrome deficient, and the near isogenic line MT: (MT/MT, au/au, MT/au and au/MT). We observed differentiated responses for many parameters analyzed. For example, the root dry mass accumulation and stern diameter obtained by MT/MT, MT/au and au/MT grafting were 33% and 31% higher, respectively, than those obtained by au/au. In the au/MT combination, there were greater root dry mass and total dry mass accumulations. Based on the changes in vegetative and reproductive development observed from grafting combinations between MT and the mutant au, we can conclude that phytochromes function in the control of photoassimilate partitioning between roots and stems during tomato growth.


Introduction
Plant development is strongly influenced by countless internal and external factors, such as the intensity and quality of red light; far-red ratios are signals that modulate the photoassimilate transport dynamics (Bocallandro et al., 2003;Tang & Liesche, 2017).However, this is dependent on the perception of light by specialized photoreceptors called phytochromes, which are part of a complex signal transduction network responsible for coordinating plant development in response to light conditions (Sun et al., 2005;Salisbury et al., 2007;Martínez García et al., 2010).These photoreceptors show two interconvertible relatively stable forms: one that absorbs red (R) light (~660 nm) and another that absorbs far-red (FR) light (~730 nm).For instance, under shading conditions, there is a reduction in the R/FR ratio that is perceived by the phytochromes and triggers intricate mechanisms that promote, for example, stem elongation to find better and adequate light conditions (Ballaré;Pierik, 2017;Van Gelderen et al., 2018).In fact, plants deficient in functional phytochromes exhibit exaggerated stem elongation and leaf chlorosis (Muramoto et al., 2005;Carvalho et al., 2011).This occurs mainly because the stem becomes the main sink of the plant and thus reduces the resources available for the development of roots and leaves (Casal, 2013).Currently, it is well known that the mechanisms by which phytochromes modulate the reduction in R/FR light responses involve many intricate molecular pathways in the shoot (Lee et al., 2017;Yang, Xie, Jiang, Z. Li, Huang, & L. Li, 2018).
Nevertheless, it has only recently been shown that phytochromes can accumulate in roots and modulate plant development (Warnasooriya & Montgomery, 2011;Costigan, Warnasooriya, Humphries, & Montgomery, 2011;Júnior et al., 2018;Sakuraba & Yanagisawa, 2018).However, one question remains: if phytochromes accumulate in the root, do they function in the mechanisms controlling the source-sink relationship that involves root and shoot communication?
To better understand the factors involved in root-to-shoot signaling, grafting has been an important research tool because molecular and/or biochemical changes, either endogenously or exogenously induced, in the scion can result from modifications in both the root and shoot (Gratão et al., 2015, Gaion et al., 2018).Thus, to study light signaling, especially that phytochrome-dependent, in photoassimilate partition control, it is interesting to associate the grafting technique with molecular tools, such as photomorphogenic mutants.
Therefore, in this work, we demonstrated by grafting that the presence of Micro-Tom functional phytochromes and their combination, as scion or rootstock, in phytochrome-defective aurea mutant modulates the partition and allocation of photoassimilates, especially between roots and stems.

Plant Material
Seeds of tomato cv.Micro-Tom (MT) and photomorphogenic mutants deficient in phytochrome aurea (au) (Carvalho et al., 2011) were germinated in plastic trays containing a mixture of Bioplant® substrate and expanded vermiculite in a ratio of 1:1 (v:v), and 15 days after sowing (DAS), seedlings of homogeneous developmental pattern were selected.

Grafting and Cultivation
The grafted plants were obtained by cutting them into a wedge shape and inserting them into a "V"-shaped incision in the rootstock (Peres et al., 2005).A completely randomized design was used with four treatments from combinations between MT and au (MT/au, au/MT, MT/MT and au/au, the first genotype being the graft and the second the rootstock) with three repetitions.After grafting, the plants were transferred to a floating moist chamber, where they remained until 30 DAS, when the complete healing of the grafting region was verified.After this time, the seedlings were transplanted into 1.4 L pots containing the same substrate for cultivation of the abovementioned seedlings.In addition, supplemental fertilization with 1 g L -1 NPK 10:10:10 was carried out together with 4 g L -1 dolomitic limestone.Throughout the experimental period of 65 days (sowing to harvest), the plants were irrigated daily.

Parameters Evaluated
At the end of the experiment, plant growth analyses were performed, such as length, density and root area, by coloring the root system with methylene blue solution for approximately 2 minutes, followed by image recording using a Hewlett Packard digitizer model 5C and processing by Delta-T Scan software.The total leaf area was also obtained by scanning the leaves and subsequent analysis of the images by the Delta-T Devices LTD image analysis system.The length and diameter of the stem were measured using a digital pachymeter, and these measurements were made from the region of stem insertion in the soil.Finally, dry biomass accumulation was also analyzed, for which the root, stem and leaf material were separately packed in paper bags and oven-dried at 55 °C for 96 hours and weight was measured using a digital analytical balance (Denver Instrument Company AA-200) with an accuracy of 0.0001 g.In addition, the reproductive characteristics, including the number of fruits per plant, fruit diameter (using a digital pachymeter), fruit weight and fruit soluble solids content, were evaluated by means of a digital refractometer (Atago PR-101 Palette, AOAC 1997).

Statistical Analyzes
The results were submitted to analysis of variance (ANOVA) and F test, and when significant, the averages were compared using the Tukey test at 5% probability using the SISVAR ® SOFTWARE (Ferreira, 2011).

Root Development
Regarding root length, the highest values were observed in self-grafted MT/MT plants compared to au/au, MT/au and au/MT plants (Table 1).Indeed, the reduction in root growth in au/au and MT/au was expected because the synthesis chromophore of phytochrome in the roots is necessary for its photoregulation.The au mutant is deficient in this biosynthesis; consequently, root growth and development will be impaired (Muramoto et al., 2005;Costigan, Warnasooriya, Humphries, & Montgomery, 2011;Bianchetti et al., 2017).However, the root length of au/MT also decreased when compared to MT/MT.
Similarly, root density (Table 1) was significantly higher in the self-grafted MT/MT (18.38 mg cm -3 ) than in the au/au (11.83 mg cm -3 ) and au/MT (12.28 mg cm -3 ), but no difference was observed when compared to MT/au (14.45 mg cm -3 ).In fact, several studies have evidenced the involvement of phytochromes as part of the mechanisms that modulate the production and allocation of photoassimilates from shoots to roots to promote the latter's development (Kasperbauer & Hunt, 1992;Wang et al., 2016;Tang & Liesche, 2017).
However, our results demonstrate that, in addition to modulating development, phytochromes act in partitioning and mobilization of photoassimilates to roots, for instance, the accumulation of RDM (root dry mass) obtained in the grafting of MT/MT, MT/au and au/MT was 33% higher than that in au/au.Therefore, considering the higher accumulation of biomass in au/MT (0.03 g plant -1 ) than in au/au (0.02 g plant -1 ), it is possible to infer that the action of phytochromes reflected positively on the root dry mass accumulation.
Thus, we determined that the light signal perceived by the grafting combination that has perfect functional phytochromes in shoots or roots (MT/MT, MT/au and au/MT) was responsible for the greater content of photoassimilates; these photoassimilates produced in shoot were reallocated to roots, favoring root growth mainly from MT/MT and MT/au combinations.

Shoot Development and Dry Mass Accumulation and Partitioning
To better understand the role of root phytochromes in shoot development, biometric analyses were performed measuring the following: stem diameter (SD), stem elongation (SE), leaf area (LF), dry stem mass accumulation (DSMA) and leaf dry mass (LDM) (Table 2).Initially, we observed that SE from MT/MT, MT/au and au/MT was very similar.On the other hand, when we compared MT/MT stem elongation with the mutant defective in phytochrome self-grafted au/au plants, a 31% reduction in stem elongation was observed (Table 2).Indeed, it is known that the deficiency in phytochromes present in au induces greater stem elongation, resulting in diameter reduction (Terry & Kendrick, 1996;Carvalho et al., 2011), which is consistent with our results (Figure 1).
Thus, SD is an important trait to be evaluated because it is directly related to stem growth and consequently to photoassimilate accumulation in this organ (Casal, 2013).In this direction, what has been observed in this study was as follows: although there was a considerable reduction in au/au SD compared to MT/MT, the SE was not significantly affected (Table 2).Moreover, it is important to note that this parameter was slightly higher in the au/au (10.80 cm) and au/MT (10.0 cm) plants than in MT/MT (9.89 cm) and MT/au (9.31 cm).------------- Note.CV; coefficient of variation.Means followed by distinct letters in the columns, differ by Tukey test (P < 0.05).**; and ns: significant (P < 0.01); and not significant, respectively.
In fact, in addition to an inverse relationship between size and soluble solids content, fruits constitute a strong draining organ that demands reserves of other parts of the plant during the reproductive period (Rodrigues et al., 2014;Turhan, Ozmen, Serbeci, & Seniz, 2011).Therefore, there are many mechanisms involved in the import, metabolism and accumulation of sugars in tomato fruits, influenced directly by a complex signaling pathway between phytochromes and hormones (Gupta et al., 2014;Bianchetti et al., 2017).However, our results indicate that the phytochrome-dependent responses observed in the au mutation are triggers near the reproductive stage, suggesting that they can synthesize some amount of functional phytochrome at this stage, even maintaining its characteristic phenotype with chlorotic leaves throughout the cycle (Sharrock, Parks, Koornneef, & Quail, 1988;Tuinen et al., 1996;Terry, Ryberg, Raitt, & Page, 2001).
In summary, our results provide information about the control of the partition of photoassimilates and their relocation of the source organs to the sink, which in part is controlled by accumulated in the roots, involving complex morphophysiological differentiation that still needs to be further explored in au as well as in its grafting combinations with MT.

Conclusion
Based on the changes in vegetative and reproductive development observed from the grafting combinations between MT and the mutant au, we concluded that phytochromes function in the control of the partition of photoassimilates between roots and stem during the growth of the tomato.

Table 1 .
Length, density, root area and dry mass of auto-grafting plants of MT and au and their reciprocal combinations MT/au, au/MT

Table 3 .
Total fruit, fruit diameter, fresh fruit weight and soluble solids contents in the fruits of the auto-grafting MT and au and their combinations MT/au, au/MT