Leaf proline content and its relation to fruit load and flowering in citrus under field conditions Conteúdo de prolina foliar e sua relação com a carga de frutos e floração em citros sob condições de campo

- Citrus species grown in temperate zones have sprouting inhibited in winter and this is retaken in spring when they also bloom. The main factor that defines the vegetative or reproductive destiny of the buds is the presence of fruits in development. Low winter temperatures slow plant metabolism, which has been reported as necessary for the expression of the reproductive program. During the winter period of the annual cycle, osmotically active compounds such as proline are increased as part of the cold tolerance response. We investigated whether the presence of developing fruit and the application of exogenous gibberellins affect the proline level in citrus leaves. In an orchard of adult ‘Montenegrina’ mandarin trees, leaf proline contents were measured over two rest periods for branches with or without fruits. Branches that bloomed, the ones that did not have developing fruits, had higher proline levels as well as higher proline: chlorophyll and proline: total amino acid ratios than did branches with fruits. However, the application of exogenous gibberellins, which reduced flowering, did not affect proline content during the same periods. We discuss the ways in which proline may be involved in floral induction in citrus.


Introduction
Knowledge of the floral induction process is the key to understanding the reproductive cycles of woody polycarpic species. The effective management of fruit crops requires knowledge of the factors that determine the productive potential of the following annual cycle. In many fruit species, the presence of developing fruits is a main factor that negatively affects yield potential of the following year, process known as alternate bearing. In citrus, the number of developing fruits and the time they remain on the plant determine the degree of flowering inhibition in the following spring (VERREYNNE and LOVATT, 2009;MARTÍNEZ-FUENTES et al., 2010;DAVEMPORT 1990;SAMACH and SMITH, 2013). Fruit is a powerful sink during its development and limits the development of vegetative structures and roots as well as the plant's ability to accumulate reserves. When there are no fruits in development, photosynthesis in the canopy is reduced. However, environmental factors may limit the increase in the net assimilation rate in response to the demand of the developing fruit (SYRVERTSEN et al., 2003, RIBEIRO et al., 2009NEUBAUER et al., 2011).
Gibberellin is a well-known endogenous factor to be strongly and negatively correlated with flowering levels in citrus (GOREN and GOLDSCHMIDT, 1970;GRAVINA, 2007;IGLESIAS et al., 2007;KOSHITA et al., 1999). Several studies show that the foliar application of gibberellic acid during the autumn-winter rest period reduces flowering intensity in citrus (GUARDIOLA et al., 1982;MUÑOZ-FOMBUENA et al., 2012). Genetic studies also support the negative effects of developing fruit (SHLIZERMAN et al., 2012) and exogenous gibberellins (GOLDBERG-MOELLER et al., 2013;MUÑOZ-FAMBUENA et al., 2012) on Cs FT2 gene expression. Based on these findings, various approaches to remove fruit from the branches have been investigated to evaluate their effects on floral induction.
Regarding to environmental factors, low temperature and water stress each induce flowering in citrus by increasing CiFT expression (NISHIKAWA et al., 2007;CHICA and ALBRIGO, 2013). Furthermore, the process of acclimatization (developing hardiness) in citrus, which begins in the winter in response to low temperature, is accompanied by increases in proline (Pro) levels (KUSHAD and YELENOSKY, 1987). Increases of this amino acid have also been observed as a response to water stress (GARCIA SANCHEZ et al., 2007;RABE et al., 1990;ZAHER-ARA et al., 2016). Many studies have investigated the role of proline as an osmolite in the plant response to stress (SZABADOS and SAVOURE 2009;SIGNORELLI, 2016). An increase in Pro level protects the plant from stress but can also occur as part of a sequence of signals to modify plant development (MAGGIO et al., 2002;KAVI KISHOR et al., 2015). Pro has also been reported to influence flowering transition in other species (MATTIOLI et al, 2009;SAMACH and SMITH 2013;ZDUNEK-ZASTOCKA et al., 2017).
Nitrogen metabolism is involved in the regulation of the annual cycle of woody species (MILLARD and GRELET, 2010). In citrus, the levels of arginine and aliphatic polyamines are related to flowering (ALI and LOVATT, 1995;LOVATT et al., 1988;SAGEE and LOVATT 1991;MARTINEZ-FUENTES et al., 2010 SYVERTSEN andSMITH, 1983). It has also been postulated that the depletion of carbon skeletons can lead to the accumulation of low-molecular weight nitrogen compounds, involving the carbohydrate budget as an incident factor in citrus flower induction (RABE, 1990). Current approaches have shown that the remobilization of C and N reserves are regulated processes of the general proteins turnover in which the proline constitutes a signal molecule (ZDUNEK-ZASTOCKA et al., 2017) and the concept that stress conditions for plants constitute inductive conditions of flowering is accepted (TROVATO et al., 2008;TAKENO 2016;SINGH et al., 2017). The process of signal transduction in which proline participates in reproductive development and its cross talk with stress is unknown. No reports have been found in citrus.
There is substantial research on the hindering effects of developing fruit and gibberellic acid application on flowering induction in citrus. However, research that links these factors with nitrogen metabolism regarding to citrus flowering is scarce. In our study, we select a mandarin with great sensitivity to alternate bearing and late harvest, which implies a strong fruit hegemony. 'Montenegrina' mandarin is a mutation of Mediterranean or willowleaf mandarin (Citrus deliciosa Tenore), (SCHÄFER et al., 2001).
The aim of this study was to determine whether leaf Pro content during the winter rest period is related to spring bloom intensity by comparing bearing and non-bearing branches with and without foliar gibberellin application.

Materials and methods
The study was conducted in a commercial orchard of seven-year-old 'Montenegrina' mandarin trees (C. deliciosa Tenore) grafted on 'Carrizo' citrange rootstocks (Poncirus trifoliata L. Raf. x C. sinensis L. Osb). The orchard was located in Kiyú (34°39'S, 56°48' W), San José, Uruguay, and the trees were cultivated under drip irrigation. In Year 1, a complete block design was used with 3 replications where each tree was considered as a block. The experimental plot consisted of two similar individual branches: one used to collect leaf samples, and one used to measure bloom intensity. Branches with a diameter of 2 cm in the base were selected, of medium Rev. Bras. Frutic., Jaboticabal, 2019, v. 41, n. 2: (e-087) vigor and without biotic or abiotic damage, which had sufficient annual shoots to account for at least 100 total nodes. Two treatments were evaluated: 1) branches with fruits, and 2) branches without fruits. Given the significant results of the presence of fruits on proline leaf levels in the first year, in the second year, the known negative effect of gibberellic acid winter sprays on flowering was included as a new factor. The effect of gibberellic acid depends on time application, so four different application dates were included as four new treatments. For these reasons, in Year II, a balanced incomplete block design was used. Each tree was an incomplete block, to which two of the six treatments were applied. The experimental plot was the same as in Year I. Each treatment had 3 replications, resulting in a total of 45 trees. The six treatments were: 1) branches with fruits, 2) branches without fruits, 3) branches without fruits + Gibberellic acid (GA 3 ), on May 23 rd , 4) branches without fruits + GA 3 on June 6 th , 5) branches without fruits + GA 3 on June 20 th , and 6) branches without fruits + GA 3 on July 11 th . The absence of fruit was achieved by manual thinning prior to the end of the physiological fruit drop period), stage 74 BBCH Scale (AGUSTI et al., 1997). The fruit-bearing branches had between three and six fruits per 100 nodes. GA 3 application was at 100 mg.L -1 on each date, and the GA 3 solution was acidified to pH 4-4.5 with phosphoric acid according to GREENBERG and GOLDSCHMIDT (1989). The GA 3 solution was sprayed locally on the branch, using a manual sprayer with a non-ionic wetting agent at a rate of 2.5% v/v.

Flowering evaluation
Branches including the last three flushes of growth were tagged to measure bloom. On these branches, we counted the total number of flowers and the total number of nodes, in order to eliminate the effect of the different number of nodes between branches and work with the variable flowers per 100 nodes (MARTINEZ-FUENTES et al., 2010). The intensity of flowering was measured in full bloom, stage 65 of BBCH scale, (AGUSTI et al., 1997). Full bloom dates were October 15 th in the Year I and October 20 th in the Year II. The flowering lasted 20 days in both years

Proline, Amino Acid and Chlorophyll Analyses
Proline, total amino acid (AA) and total chlorophyll (Chlo) contents were measured in tissue discs collected from adult leaves with a fresh weight (FW) of at least 0.5 g, and the contents were expressed as µmol.g -1 FW. Three repetitions per treatment per date were taken. Following their collection, the leaf discs were immediately frozen in liquid nitrogen in the field and were maintained at -80°C until processing. Sampling was conducted during the winter rest period (April to July) every two weeks for two consecutive years ('off' and 'on' years). For the GA 3 application treatments, Pro content was measured only fourteen days after application. Borsani et al. (1999) described the extraction and quantification methods for Pro and AA. Chlo was measured according to the Wellburn (1994) protocol. The three compounds were expressed in fresh base, and the Pro/Chlo and Pro/AA ratios were calculated.

Statistical Analysis
Linear mixed models were used for all analyses. In Year I, the complete blocks were treated as fixed effects. For the two treatments considered, time-course of the quantified leaf metabolites was evaluated with a repeated measures analysis to analyze the main effects (treatment and date) and their interaction.
In the second year, although a partially balanced design that included 6 treatments was used, only treatments one and two were evaluated in all dates during the winter rest period, so the data was analyzed in two ways. First, only treatments 1 and 2 were considered, therefore only the three trees that had both treatments were included (i.e. each treatment was repeated three times), and data for this two treatments was analyzed as if it was a randomized complete block design. For these two treatments, a repeated measures analysis was done, as in the first year. In a second approach, treatments were compared within each date, since in each sampling date only treatments 1 and 2 and the corresponding treatment with GA 3 that had been applied 14 days before were evaluated. In this case, the incomplete blocks were treated as random effects, nested in each complete replication. Mean comparisons of flowering intensity for both years and of metabolite contents of leaves for 'on' years were performed with Tukey tests (P<0.05). All of the analyses were performed with SAS [Version 9.2] (SAS Institute, 2005).

Results and discussion
The results confirmed the inhibitory effects of fruit presence in both years, and the application of gibberellic acid (GA 3 ) during winter rest period in Year II, on subsequent spring flowering (GARCÍA-LUIS et al., 1986;ZHANG et al., 2011) (Table 1 and 2). The magnitude of flowering inhibition by fruit was similar to that reported in studies of other mandarin cultivars (MUÑOZ-FAMBUENA et al., 2011;VERREYNNE and LOVATT 2009, ( (Table 2). Thus, the present results validate the field experimental strategy of this study.
The proline level in leaves of 'Montenegrina' mandarin during the winter varied between minimum and maximums values of approximately 17 and 45 µmol.g -1 FW in Year I and between 30 and 65 µmol.g -1 FW in Year II (Figure 1 and Table 3). These values are similar to those reported for various tissues from citrus species (MADEMBA-SY et al., 2003;MORENO and GARCÍA MARTÍNEZ, 1983;KATO, 1986;SHRAMA et al., 2011). Differences between years can be expected considering that the present study was a field study and that many environmental factors determine plant ability to produce this amino acid (KAVI KISHOR, et al., 2015). The highest proline levels measured in Year II can be associated with a greater accumulation of Chilling Hours during the winter compared to Year I (Figure 4) as the acclimation response reported by KUSHAD and YELENOSKY (1987).
The analysis of repeated measures of Pro content and their relations with Chlo and totals AA measured on the leaf based on data from treatment 1 and 2, showed no significant interaction of treatment and sampling date (p-value>0.05). Only treatment main effect was significant on average Pro, Pro/Chlo and Pro/AA levels (p-value <0.05).
Pro levels were higher in leaves sampled from branches without developing fruits than in those from fruit-bearing branches in both studied years. Branches with fruits had 30 and 39% less Pro than did the branches without fruits in Year I and II, respectively (Table 1). Table 3 shows comparisons of proline averages by date of sampling, in the 6 treatments of Year II. Exogenous GA 3 did not modify proline levels measured 14 days after its application in any of the dates despite having modified flowering intensity. Leaf proline contents of the branches without fruit treated with GA 3 were equal to the leaves of the branches without fruit and without hormone spray. No reports were found that correlate the proline synthesis with the gibberellin signalling that allow us to explain the results obtained.
Chlo content was not affected by treatments and sampling date, which indicated stability in the photosynthetic structures. Total AA content was significantly affected by sampling date but not by fruit presence with p values of 0.0001 and 0.5003 respectively.
Pro/AA and Pro:Chlo ratios were significantly affected by developing fruit, being higher in leaves from shoots without fruits (Table 1 and Figure 2 and 3). The findings that a high Pro:AA ratio was maintained during the study period and that no effect of treatment on AA content was observed suggest that the detected Pro did not come from proteolysis. De novo synthesis of Pro has been reported in response to drought (BORSANI et al., 1999;DÍAZ et al., 2005). If the observed Pro was the result of proteolysis, then the amino acid contents should have also increased. However, studies of the enzymatic activity are necessary to elucidate to what extent the proline contents in tissues and plants are regulated by biosynthesis, degradation and intra as well as intercellular transport processes (LEHMANN et al., 2010). The proportion of Pro with respect to the total amino acids was higher than 50% in all cases, which is higher than the values reported by MADEMBA-SY et al. (2003).
Analyzing the time course of the variables studied from date to date, we can observe that the differences between treatments disappeared at the end of the study period for Pro ( Figure 1) and Pro:AA ratio in Year I ( Figure 3) and in both years for Pro:Chlo (Figure 2). However, the period between May 4 th and July 6 th for Year I and the period from May 25 th to July 11 th in Year II, the variables were always higher in the leaves of the fruitless branches. Within that period, there was a tendency to reduce values with average reductions between 22% and 42% for Pro, 11% and 54 % for Pro:Chlo and 13 and 36% for Pro:AA as difference between maximum and minimum for both treatments. At the end of winter and specifically in mid-July in our conditions, the increase in temperatures determine the release of the ecodormancy. Then, the differentiation process of the meristems in new floral and / or vegetative structures, and changes in the C and N metabolism begins. Difference between thermal regimes and annual radiation explain the variations in the beginning of the sprouting and flowering (MICHELOUD et al., 2018).
In summary, we conclude that the level of flowering in the spring is positively correlated with leaf Pro content in the previous winter. Higher levels of Pro in the leaves during the winter were found for branches that did not bear fruit and had higher flowering levels in the following spring than for fruit-bearing branches. These results demonstrate that Pro is involved in floral induction in citrus, as has been reported in other species (MATTIOLI et al, 2009;SAMACH and SMITH 2013;ZHANG et al., 2015;SINGH et al., 2017). If Pro is part of the chain of signals that allow floral expression in citrus, we can conclude that its effect in this study occurred prior to the negative effect of exogenous gibberellins or independently, which inhibited flowering but did not modify Pro level.
A question that arises from these conclusions is through which mechanism the absence of developing fruits in branches increases the synthesis of Pro. Possible ways to approach this question include studying the processes of energy dissipation in photosynthesis, the loading and unloading of phloem, or in contemporary sinks and Pro metabolism. DIAZ et al. (2005) reported increases in Pro synthesis when there was an excess of reducing power that was not used in the carboxyl phase of the photosynthesis. The absence of developing fruits determines increases in the contents of starch in the leaves (MARTINEZ-FUENTES et al., 2010), a process that Rev. Bras. Frutic., Jaboticabal, 2019, v. 41, n. 2: (e-087) negatively regulates the net assimilation of carbon. In these conditions, excesses of reducing power and energy of the photochemical phase of photosynthesis could induce a change in the proline synthesis (DIAZ et al., 2005).
The relationship C / N has been reported as a factor linked to the expression of reproductive programs in annual species (CORBESIER et al., 2002), however, the way in which both Carbon and Nitrogen are quantified and their fractions differ and have not been considered. In citrus, it has been proposed (LOVATT and MARTINEZ) that high levels of N as nitrate or ammonium in the leaves, are associated with a high flowering as well as levels of polyamines. RABE (1990) postulates the absence of carbon skeletons as a condition for the increase of nitrogenous compounds of low molecular weight since the floral induction in citrus responds to stress, water or low temperatures. In our results, it was verified that the presence of fruit was imposed on the inductor effect of cold winter (stress), very similar between the years of the study. Therefore, the absence of sink and the inhibition of photosynthesis could be another condition of stress, not so the insufficiency of carbon skeletons. Whatever the mechanism by which the fruit inhibits floral induction involving proline, our results add to the scant evidence that N metabolism is altered during the floral induction period. Proline was reported as a signal compound in reproductive processes and its synthesis is increased under stress conditions. Therefore, it is feasible that it constitutes a link between floral inductions at the time citrus perceive low temperatures, being also regulated by the presence of fruits in development. Further studies with this approach should be carried out considering genetic evidence of the environmental regulation of the annual cycle of woody polycarpic species, (TAN AND SWAIN, 2006, GYLLENSTRAND et al., 2007, NISHIKAWA et al., 2007. The opposition between vegetative and reproductive development is the result of the plant's ability to adapt to the environment (HANKE et al., 2007). Under this optics, we could possibly think about a change in the partition of assimilated and / or N, due to the presence of fruits in the branches, as a regulating process of floral expression.
Although more evidence is needed to clarify the role of Pro in citrus floral induction, this study provides evidence that fruit effects on the flowering induction process involve changes in leaf Pro content during the winter rest period.    Mean and standard error of proline leaf content (µm.g -1 FW), proline:chlorophyll ratio (Pro:Chlo), proline:amino acid ratio (Pro:AA) and flowers per 100 nodes in the following spring for branches with fruits (+ fruit) and branches without fruits (-fruit  10.20 b Branches -fruit + GA3 20 June (T5) 9.10 b Branches -fruit + GA3 11 July (T6) 9.40 b *Different letters in the same column indicate significant differences (P<0.05)

Conclusions
Although more evidence is needed to clarify the role of Pro in citrus floral induction, this study provides evidence that fruit effects on the flowering process involve changes in leaf Pro content during the winter rest period. This relationship is independent of the inhibitory effect of exogenous gibberellins on citrus flowering.