Mature Citrus Leaves Undergo Coordinated Photosynthetic Downregulation to Support Flush-Driven Carbon and Nitrogen Sink Demand

31 The source /i1 sink attenuation hypothesis suggests that plants regulate carbon fixation in 32 response to fluctuations in sink demands. Many evergreen trees exhibit flushing growth 33 patterns, where new shoot development generates a strong, transient demand for both carbon 34 and nitrogen that may influence the function of mature leaves. This study examined the 35 source–sink attenuation hypothesis in the context of vegetative sink growth by investigating 36 the photosynthetic capacity and nitrogen dynamics in mature citrus leaves across three stages 37 of flush development. In contrast to expectations, photosynthesis declined as flush growth 38 progressed. Early flush initiation induced stomatal limitation in mature leaves, whereas as 39 sink demand from further shoot growth continued carboxylation capacity and Rubisco 40 abundance declined, despite relatively stable total leaf nitrogen. These results suggest that 41 mature leaves undergo selective protein retooling under prolonged sink demand, constraining 42 CO/i1 fixation while maintaining C export. Overall, this study revealed that under strong 43 combined N and C sink demands, mature citrus leaves function primarily as regulated carbon 44 conduits rather than dynamically upregulating photosynthesis, providing new insight into 45 source–sink coordination in woody perennial species. 46 47

Source /i1 sink dynamics; Carbon export; Flush phenology; Leaf position; 48 Photosynthetic regulation 49 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 4

50 Perennial plants play a vital role in ecosystem and biosphere functions by contributing to 51 carbon (C) sequestration, nutrient cycling, and soil health maintenance (Van Driesche et al., 52 2010; Werling et al. , 2014). However, optimizing their productivity remains a challenge 53 because of the complex interplay between CO 2 fixation, growth phenology, and nutrient 54 availability, among other environmental factors. In agriculture, where perennial crops are 55 increasingly valued for both their ecological and economic benefits, understanding how 56 photosynthetic carbon is produced and allocated across tissues is critical for improving 57 resource use efficiency (Lamichhane and Alletto, 2022). 58 C and nitrogen (N) regulation in plants is primarily mediated by source /i1 sink interactions 59 (Lemoine et al., 2013; Paul and Foyer, 2001; Poorter et al., 2012). Mature leaves serve as the 60 primary sources of assimilates, whereas developing organs, including young leaves, roots, 61 seeds, and meristems, act as dynamic sinks (Millard and Grelet, 2010; Osorio et al. , 2014; 62 Smith et al. , 2018; Xiao-Li et al. , 2022). In evergreen perennials, repeated cycles of 63 vegetative flushes create dynamic changes in sink strength, altering the demand for exported 64 photoassimilates and potentially triggering feedback responses in source leaves (Chapin III et 65 al., 1986; Richardson et al. , 2013). Importantly, C fixation, N assimilation, and protein 66 turnover are increasingly viewed as coordinated processes that plants dynamically adjust to 67 balance growth, resource-use efficiency, and developmental demand, rather than maintaining 68 maximal photosynthetic capacity at all times (Liu et al., 2025). 69 While numerous studies have addressed the effects of leaf age and position on photosynthetic 70 capacity, the findings remain inconsistent (Gonzalez /i1 Real and Baille, 2000; Henning et al. , 71 1979; Kitajima et al., 2002). Some studies report a decline in photosynthesis with increasing 72 leaf age due to reallocation of N or reduced sink strength (Crous et al., 2021; Menezes et al., 73 2022; Xie and Luo, 2003), whereas others observe sustained or even enhanced photosynthetic 74 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 5 capacity in mature leaves (Frank, 1981; Proietti et al. , 2000). Moreover, the role of leaf 75 position in mediating source strength and carbon export remains underexplored. 76 Citrus trees display a growth habit that is useful in studying source /i1 sink dynamics. Citrus 77 trees exhibit a flushing phenology in which shoots emerge with all leaf primordia and 78 complete their growth in approximately three weeks (Syvertsen, 1985). The pattern of 79 phenology involves multiple cycles per year in which newly matured shoots are quiescent (3-80 4 weeks), followed by periods of rapid shoot growth (3 weeks), providing an ideal system for 81 investigating C dynamics and resource allocation in perennials. The mature leaves subtending 82 the new shoots are still in the early period of their lifespan, given that the next flush typically 83 occurs at 1-2 months and that the citrus leaf lifespan is greater than 17 months in the field 84 (Wallace et al., 1954). 85 Recent work by Li and Vincent (2022) demonstrated that the emergence of new shoots 86 enhances carbon export from mature leaves, whereas work by Kriedemann (1970) showed 87 that basal leaves tend to supply C to roots and stems (basipetal translocation), whereas apical 88 leaves preferentially support new shoot growth (apical translocation). In the case in which 89 roots are sinks, Li et al. (2024) reported increased photosynthesis in response to defoliation, 90 which increased the relative sink demand per leaf, although leaf export and stem sugar 91 transport speeds did not change. Subsequently, Vincent et al. (2025) showed that apical 92 leaves accumulate more starch than basal leaves when root growth is the only sink, and 93 eventually deplete nearly all starch reserves in support of new shoots growing at the apex. 94 Given that flushing increased leaf C export, on the basis of the source–sink attenuation 95 hypothesis, we expected mature citrus leaves to respond to new shoot growth with increased 96 CO2 assimilation. However, Wallace (1954) reported that mature leaves lost 25–30% of 97 soluble N to new shoot growth. Thus, while providing a strong C sink, new shoots may also 98 be a strong N sink. Given that leaf position impacts C export and allocation in response to the 99 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 6 strong C and N sink dynamics imposed by new shoot growth, we conducted a study to assess 100 C fixation and transport dynamics in response to flushing. 101 We hypothesized that (1) the emergence and development of new shoot flushes increase sink 102 demand, which in turn stimulates both C export and photosynthesis in mature source leaves, 103 and (2) apical and basal leaves differ in their physiological responses, reflecting their distinct 104 roles in C allocation. To address these hypotheses, we studied mature citrus leaves across the 105 course of vegetative phenological cycles and used gas exchange to assess their photosynthetic 106 capacity and activity; 14C labeling to address C transport; and various methods to assess N, 107 chlorophyll, and Rubisco contents. The results offer new insights into how source leaves 108 integrate internal sink signals to adjust C metabolism, thereby advancing our understanding 109 of source–sink regulation in citrus and other evergreen woody perennials. 110 111

112 Plant material and growth conditions 113 In November 2022, healthy sweet orange plants (C. sinensis [L.] Osbeck), cv. Valencia 1-14-114 19, which were grafted onto ‘US-942’ rootstock ( C. reticulata ‘Sunki’ x Poncirus trifoliata 115 ‘Flying Dragon’), were sourced from a commercial nursery. The plants were acclimated in a 116 controlled greenhouse environment at the University of Florida, Citrus Research and 117 Education Center in Lake Alfred, FL, USA (28.1021° N, 81.7121° W). The plants were 118 subsequently transplanted into 10-L containers filled with a ‘Pro-mix BX’ mixture (Premier 119 Tech Ltd., Quebec, Canada) to ensure uniform growth conditions. In February 2023, the 120 plants underwent pruning to promote uniformity, with each plant pruned to maintain 4–5 121 evenly distributed branches along the main stem. New shoots began to emerge 7–10 days 122 post-pruning, and the number of shoots was controlled for consistency across the plants. For 123 experimentation, one branch per plant was selected, featuring 8–9 fully expanded leaves on 124 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 7 mature flushes (Table S1) and measuring approximately 14–15 cm in length. Once mature, 125 these branches were used for all measurements over the subsequent flushing cycle. 126 Throughout the experiment, the greenhouse temperature was maintained between 24°C and 127 35°C, with the relative humidity ranging from 64% to 76%. The temperature fluctuations 128 were controlled by a pad and fan system, which was automatically triggered when the 129 temperature reached 30°C. Fertilization was provided biweekly, with 2.5 g of 20-20-20 130 fertilizer applied to the surface of each pot. Weekly pest inspections were conducted 131 following the guidelines outlined in the citrus management guide (Diepenbrock et al., 2019). 132 Experimental Design 133 This study employed a three-stage experimental design to investigate the effects of 134 phenological stage on C dynamics in sweet orange plants (Fig. S1). The three stages were as 135 follows: mature branches only (stage 1), mature branches with the initiation of new shoot 136 growth (stage 2), and mature branches with fully expanded new shoots (stage 3). 137 At each stage, four trees were selected for analysis. Gas exchange measurements and C 138 labeling experiments were performed on both the apical and basal leaves of the same branch. 139 All selected leaves were healthy and fully expanded, ensuring consistency across the 140 experimental setup. 141 Branch Length and Leaf Count 142 Branch length was measured using a tape measure, and the number of leaves on each branch 143 was manually counted. These data were used to characterize the branches and ensure 144 uniformity across the experimental stages. 145 Gas exchange measurement 146 Gas exchange parameters were measured using an infrared gas analyzer (LI-6800, LI-COR 147 Biosciences, Lincoln, NE, USA) to determine the net assimilation rate ( A), transpiration rate 148 (E), intercellular CO2 concentration (Ci), and stomatal conductance (gsw). Measurements were 149 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 8 conducted between 09:00 a.m. and 11:00 a.m. under steady light intensity (1200 µmol m-2 s-1) 150 and a CO2 concentration of 400 µmol mol-1. 151 Dynamic Assimilation CO2 Response Curves 152 Dynamic assimilation CO2 response curves were generated using the LI-6800, following the 153 procedure described by Saathoff and Welles (2021). The A /Ci response curves, which assess 154 net CO 2 assimilation under saturating light to intercellular CO 2 concentrations, were 155 generated with the following parameters: a water vapor mole fraction of 25 mmol H2O mol-1, 156 a photosynthetic photon flux density of 1400 µmol m -2 s-1, and a constant flow rate of 300 157 µmol s -1. The CO 2 concentration started at 100 µmol mol -1 min-1 and increased at a rate of 158 100 µmol mol-1 min-1 until it reached 1400 µmol mol-1. 159 Estimation of Photosynthetic Parameters 160 The raw RACiR data were processed using the racir package (Stinziano et al., 2019) to create 161 a calibrated A/Ci curve. The function fitaci() from the plantecophys package (Duursma, 2015) 162 was used to estimate photosynthetic parameters, including the Vcmax, J1400, TPU, and two 163 transition points: the transition from carboxylation to electron transport rate limitation states 164 (Citrans1) and the transition from the electron transport rate to triose phosphate utilization 165

states ( Citrans2). Additionally, stomatal limitation to photosynthesis was calculated 166 by comparing the value of A for each leaf to what value of A would be if C a = Ci (e.g., Ci = 167 400 ppm) based on the A/Ci response curves. 168 Chlorophyll measurement 169 Leaf discs were placed in 2 mL Eppendorf tubes with 1 mL of DMSO and stored in the dark 170 at room temperature. The next day, the solvent was transferred to a new tube, and another 1 171 mL of DMSO was added to the leaf discs. After 3–4 days, the pigments were fully extracted, 172 turning the solvent green and the discs translucent. The solution was mixed, and 200 μ L was 173 pipetted into a well plate with DMSO as the blank. The absorbances at 665 nm, 649 nm, and 174 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 9 480 nm were measured using a spectrophotometer. The chlorophyll content was calculated 175 via the following equation: 176 Chla+b [μ g] = [(A665 - A700) * 8.02 + (A648 - A700) * 20.2] * S 177 Chla+b [μ g g-1] = Chla+b [μ g]/FW 178 where A 665 is the absorbance at 665 nm, A 648 is the absorbance at 648 nm, A 700 is the 179 absorbance at 700 nm, S is the amount of solvent used, and FW is the fresh weight of the 180 sample. 181 Nitrogen content analysis 182 The leaf samples were dried in a forced-air oven at 55–60°C for 72 hours to remove excess 183 moisture. The dried samples were then ground into a fine powder using a ball mill to ensure a 184 uniform particle size. Each powdered sample (100 mg) was subsequently analyzed on a Leco 185 CN828 Carbon and Nitrogen Analyzer (Leco Corporation, St. Joseph, MI, USA) to determine 186 the N content. 187 Rubisco Quantification 188 The Rubisco content in the leaf tissues was measured using a competitive ELISA. Fresh leaf 189 samples were ground in liquid nitrogen with a mortar and pestle, and proteins were 190 subsequently extracted using the 1× sample extraction buffer supplied with the Rubisco 191 ELISA Kit (Novus Biologicals, NBP2-60142) at a tissue-to-buffer ratio of 1:5 (w/v). The 192 extracts were immediately stored at -80/i1 °C until further analysis. 193 Quantification was performed according to the manufacturer’s instructions. Briefly, samples 194 were loaded into 96-well microplates precoated with Rubisco antigen. In this competitive 195 assay, the Rubisco sample competes with the immobilized antigen for binding to a specific 196 antibody. HRP-conjugated goat anti-rabbit IgG was added for detection. Upon addition of the 197 substrate, a blue color developed, which turned yellow after acid neutralization. The 198 absorbance was measured at 450 /i1 nm with background correction at 540 /i1 nm using a 199 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 10 FlexStation 3 spectrophotometer (Molecular Devices, USA). The final Rubisco 200 concentrations were determined by comparing the sample absorbance to a standard curve. 201 Seven calibration standards (0, 3.12, 6.25, 12.5, 25, 400, and 800 /i1μ g/mL) were prepared to 202 generate a standard curve. The relationship between the absorbance and Rubisco 203 concentration was modeled using a four-parameter logistic (4PL) regression equation. All 204 measurements were performed in triplicate across three biological replicates. Negative 205 control wells lacking samples, antibodies, and HRP conjugates were included to assess the 206

signal and ensure assay specificity. 207 Statistical analysis 208 Statistical analysis was performed using Statistix 8.1 software (Analytical Software, 209 Tallahassee, FL, USA). The data were subjected to analysis of variance (ANOVA) to 210 determine the significance of differences among the treatments and stages. When significant 211 differences were detected, the means were compared using the honestly significant difference 212 (HSD) test at a significance level of p≤ 0.05. Correlation analysis was performed in R (version 213 4.2.2, https://www.R-project.org/), using the ggpairs() command in the {Ggally}package 214 (Schloerke B et al. , 2024), which includes Pearson correlations and their P values for the 215 correlations among variables throughout the entire experiment and within the phenological 216 stage or within branch position. 217 218

219 Trend of Photosynthetic Capacity in Citrus Leaves 220 The developmental stage of the new flush (hereafter “stage”) had a highly significant effect 221 on all measured photosynthetic parameters in the mature leaves except for Citrans2 (p=0.6271), 222 whereas leaf position (apical vs. basal) influenced only TPU ( p=0.0253) and Citrans1 223 (p=0.0068) (Fig. 1). Specifically, Vcmax, J1400, and TPU all increased from stage 1 to stage 2 224 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 11 and then decreased from stage 2 to stage 3. Rd also exhibited a significant stage /i1 ×/i1 position 225 interaction: at stage /i1 2, the basal leaves maintained a greater Rd than did the apical leaves. 226 Citrans1 displayed a parallel interaction, with higher Citrans1 values in basal leaves than in apical 227 leaves at stage/i1 3 (Fig. 1). 228 Gas/i1Exchange Dynamics 229 Consistent with the changes in biochemical capacity, whole /i1 leaf gas exchange parameters 230 (E, A, Ci, gsw and gsw limitation) were strongly influenced by stage (Fig. 2). In detail, at the 231 ambient CO2 concentration, E, A, Ci, and gsw declined progressively from stage /i1 1 through 232 stage/i1 3, reflecting reduced stomatal opening and CO/i1 uptake as the flush matured. Stomatal 233

( gsw limitation) behaved differently: it rose from stage /i1 1 to stage /i1 2, indicating 234 increasing diffusion constraints as new sinks emerged and then fell sharply by stage /i1 3. Leaf 235 position had no detectable main effect or interaction effect on these gas exchange traits. 236 C Export Patterns in Citrus Leaves 237 The whole/i1 leaf 14C export rates were significantly affected by leaf position ( p=0.0056; Fig. 238 3). Basal leaves exported more C at stage 2 than at stage 1 and stage 3, whereas apical leaves 239 presented the opposite trend. Notably, at stage 2, the basal leaves exported approximately 240 82% of the newly fixed C, whereas 56% of the apical leaves did. No significant position 241 differences were observed at stages /i1 1 or /i1 3, suggesting that positional partitioning of 242 exported assimilates is most pronounced during the onset of new sink growth. 243 Chlorophyll, Nitrogen, and Rubisco Contents 244 The total chlorophyll content mirrored this pattern, increasing from stage /i1 1 to/i1 2 and then 245 decreasing at stage /i1 3 ( p = 0.0003), with basal leaves consistently maintaining higher 246 pigment levels than apical leaves did (p = 0.007; Fig./i1 3A). 247 The leaf total N content was significantly influenced by the leaf position ( p = 0.0048; Fig. 248 3B). Basal leaves contained more N than apical leaves did in stages /i1 1 and/i1 2, but basal N 249 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 12 declined by ~16% in stage /i1 3, suggesting remobilization or dilution. The Rubisco content 250 peaked at stage /i1 2 (p = 0.0306; Fig. 3C), indicating elevated photosynthetic capacity during 251 flush initiation, and then decreased by stage /i1 3. Position did not influence Rubisco 252 abundance. 253 Correlations among Physiological Parameters 254 Correlation analyses revealed several consistent relationships among photosynthetic capacity, 255 carbohydrate status, and stomatal behavior (Fig. 4). Across leaf positions, Vcmax was strongly 256 correlated with J 1400 and g sw limitation, underscoring the coordinated regulation of 257 carboxylation and electron transport with stomatal control. Importantly, Vcmax and J1400 were 258 positively correlated with Rubisco in basal leaves but not in apical leaves, suggesting that 259 Rubisco abundance may play a stronger role in determining carboxylation capacity in basal 260 leaves. This finding is consistent with the greater fluctuations in Vcmax and Rubisco observed 261 in basal leaves across stages. A was correlated with g sw at both leaf positions, reflecting the 262 central role of stomatal conductance in driving CO/i1 assimilation. 263 Stagewise analyses (Fig. 5) further supported these relationships. V cmax and J1400 were 264 consistently correlated with gsw limitation at stages 1 and 2, whereas at stage 2, both traits 265 were also correlated with Rubisco. A maintained a strong positive association with gsw at later 266 stages, indicating that stomatal regulation continued to constrain assimilation as the flush 267 progressed. Together, these patterns highlight how photosynthetic capacity and stomatal 268 behavior are interdependent, with basal leaves showing stronger coupling of Rubisco to Vcmax, 269 which is consistent with their role in nitrogen mobilization during flush development. 270 271

272 Sink Demand Does Not Enhance Photosynthesis in Mature Leaves 273 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 13 We hypothesized that the emergence of a new vegetative flush would increase sink demand, 274 leading to greater C export and an increase in photosynthesis in mature source leaves, which 275 is consistent with the source–sink attenuation hypothesis. However, our results revealed a 276 different story. Although C export increased during flush initiation (stage 2; detailed in 277 (Vincent et al. , 2025), photosynthetic activity ( A, gsw) and photosynthetic capacity ( Vcmax, 278 J1400) decreased from stage 1 to stage 3. This response contradicts the source–sink attenuation 279 model, which predicts stimulation of photosynthesis under elevated export demand. Instead, 280 our findings suggest that flush-induced sink demand exceeds the capacity of mature leaves to 281 sustain C assimilation, ultimately leading to depletion of leaf carbohydrate reserves, as 282 reported previously (Vincent et al., 2025). 283 The decline in A despite increased C exports reflects a coordinated, stage-dependent shift in 284 photosynthetic limitation. During early flush initiation (stage 2), reduced A was driven 285 primarily by a decline in g sw, whereas during later flush development (stage 3), further 286 reductions in A were associated with decreases in Vcmax and J 1400. These later declines 287 coincided with reduced Rubisco abundance and modest changes in total N. Correlation 288 analysis reinforced this interpretation, as Vcmax and J1400 were strongly associated with 289 Rubisco content, particularly in basal leaves, indicating that the degradation of photosynthetic 290 proteins directly constrained carboxylation capacity as sink demand intensified. 291 Importantly, this two-phase response, initial stomatal restriction followed by biochemical 292 downregulation, aligns closely with the metabolic and proteomic reprogramming observed in 293 our companion study (Chen et al., 2026, Under Review). This work demonstrated a broad 294 downregulation of photosynthetic and primary metabolic pathways in mature leaves during 295 flush development, alongside the mobilization of C and N reserves to support sink growth. 296 Together, these findings suggest that mature citrus leaves undergo an active functional 297 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 14 transition from C assimilation toward resource export and support rather than simply 298 responding passively to carbohydrate feedback. 299 gsw is known to be influenced by sucrose availability, which plays roles in both osmotic 300 regulation and metabolic signaling within guard cells. In our companion study, sucrose levels 301 declined sharply as sink demand increased (Vincent et al. , 2025), whereas the present study 302 revealed a positive relationship between gsw and sucrose. Together, these findings suggest 303 that sucrose limitation impairs stomatal function by restricting osmotic or metabolic signaling 304 within guard cells, thereby increasing stomatal limitation during early flush growth. Similar 305 sucrose-dependent regulation of the stomatal aperture via hexokinase-mediated signaling has 306 been reported previously (Lima et al., 2018; Medeiros et al., 2018). Moreover, citrus girdling 307 and sink manipulation experiments have shown that the photosynthetic rate can decrease 308 before carbohydrate accumulation occurs, indicating early photosynthetic downregulation in 309 response to export stress rather than classic feedback inhibition (Iglesias et al., 2002; Nebauer 310 et al., 2011). Collectively, the integrated decline in gsw, Vcmax, and Rubisco contents reflects a 311 coordinated downregulation of photosynthesis under sustained sink demand, which is 312 consistent with carbohydrate depletion and metabolic reprogramming (Vincent et al., 2025). 313 These dynamics are distinct from those of natural leaf senescence, as the leaves examined 314 here were young (2 and 3 months old) relative to the typical citrus leaf lifespan 315 (approximately 17 months) (Wallace et al., 1954). 316 317 Positional Differences in Export but not Photosynthetic Capacity 318 Although leaf position influenced export dynamics, it had a minimal effect on photosynthetic 319 parameters. During flush initiation (stage 2), the basal leaves exported a greater proportion of 320 newly fixed carbon than did the apical leaves (Vincent et al., 2025). This positional contrast 321 is consistent with prior findings in citrus, where basal leaves are more strongly connected to 322 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 15 structural sinks such as roots and stem tissues, whereas apical leaves preferentially serve local 323 growing tips (Li et al. , 2024). Despite these export differences, both the apical and basal 324 leaves presented similar decreases in A and gsw and increases in gsw limitation across the 325 phenological stages, indicating that positional differences in export did not translate into 326 differential regulation of photosynthesis. This coordination of source activity within the 327 branch occurs in response to the whole shoot sink demand. 328 The positive correlations of Vcmax and J1400 with Rubisco in basal leaves indicate stronger 329 biochemical coupling between N investment and photosynthetic capacity in those leaves than 330 in apical leaves. This relationship aligns with the greater variability in Vcmax and Rubisco 331 observed in basal leaves, which is consistent with reports of preferential N remobilization 332 from older or basal tissues (Xiong et al. , 2025). In contrast, the apical leaves presented 333 weaker correlations between Rubisco and Vcmax, suggesting that their photosynthetic 334 adjustments are less constrained by N-related biochemical limitations and more influenced by 335 local sink connectivity. Together, these findings indicate that while export is not strictly 336 determined by source capacity, the mechanisms supporting C allocation may differ by leaf 337 position: basal leaves function as long-distance suppliers linked to protein and N turnover, 338 and apical leaves serve emerging shoot sinks through carbohydrate-driven supply. This 339 spatial partitioning of function aligns with recent work by Zhao et al. (2025), who showed 340 that C allocation in citrus leaves is shaped by genotype and leaf type, likely through 341 transcriptional regulation of sugar metabolism and transport genes. While transcriptomic data 342 were not collected in the present study, our physiological findings support a model in which 343 export is differentially regulated by leaf position, superimposed on a shared pattern of 344 declining assimilation under increasing sink demand. 345 346 Nitrogen Allocation: Stability with Functional Retooling 347 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 16 N allocation may play a major role in the photosynthetic responses observed in the present 348 study. Although new vegetative flushes are strong C sinks (Li and Vincent, 2022), they are 349 also major N sinks that require rapid acquisition of amino acids and proteins to support leaf 350 expansion and metabolic activity (Wallace et al. , 1954; Xiong et al. , 2025). A recent 351 proteomic study by Xiong et al. (2024) revealed that citrus trees remobilize N from stems and 352 leaves to support new shoot growth. Under high N supply, stem reserves are the primary 353 source of new shoot N, whereas under low N supply, leaves degrade proteins, including 354 Rubisco and vegetative storage proteins (VSPs), to fuel new flushes. In the present study, 355 total leaf N remained relatively stable across phenological stages, indicating that mature 356 leaves did not experience wholesale N depletion. However, the Rubisco content decreased 357 markedly by stage 3, which coincided with reductions in Vcmax and J1400, suggesting that N 358 was selectively reallocated away from the photosynthetic machinery. Correlation analysis 359 supported this interpretation, as Vcmax and J 1400 were correlated with Rubisco content, 360 particularly in basal leaves. These relationships indicate that reductions in carboxylation 361 capacity were driven primarily by Rubisco loss rather than by changes in overall N 362 availability. The stronger coupling observed in basal leaves is consistent with their greater 363 functional role in supporting long-distance sink demand and N mobilization. 364 This pattern closely aligns with the proteomic evidence reported in our companion study 365 (Chen et al., 2026 under review ), which demonstrated the selective degradation of 366 photosynthetic proteins, including Rubisco, alongside the maintenance of total N pools. This 367 study further revealed broader remodeling of leaf protein composition, characterized by 368 downregulation of C fixation pathways and enrichment of proteins associated with N 369 mobilization and transport. Together, these findings indicate that N remobilization during 370 flush development occurs through targeted protein turnover rather than through generalized N 371 loss. 372 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 17 Importantly, the temporal sequence observed here suggests a coordinated shift in the N use 373 strategy. Stomatal limitation dominated early during flush initiation (stage 2), whereas 374 Rubisco degradation and reduced carboxylation capacity became evident only as sink 375 demand persisted into stage 3. This delay implies that N reallocation from photosynthetic 376 proteins is not an immediate response to sink activation but rather a regulated adjustment 377 once sustained sink dominance is established. Such a strategy would allow mature leaves to 378 maintain short-term assimilation while progressively prioritizing nitrogen export to 379 developing shoots. 380 Although total leaf N changed only slightly, the functional consequences of N redistribution 381 were substantial. The uncoupling of photosynthetic capacity from total N content reflects a 382 shift in N partitioning away from C assimilation and toward supporting sink growth. Similar 383 patterns have been reported in evergreen species, where the selective degradation of Rubisco 384 and other photosynthetic proteins enables N conservation while reallocating resources to 385 developing tissues (Xiong et al. , 2025). In this context, the decline in Rubisco and Vcmax 386 observed here represents a strategic retooling of mature leaves under high sink demand rather 387 than premature senescence or nutrient deficiency. 388 389

390 This study demonstrated that vegetative flush initiation in citrus plants stimulates C export 391 from mature leaves without increasing their photosynthetic capacity, providing a clear 392 counterexample to the source–sink attenuation hypothesis. Instead, sustained sink demand for 393 both C and N drives a coordinated downregulation of photosynthesis in mature leaves. 394 Decreases in A and gsw during early flush development reflect stomatal limitation associated 395 with carbohydrate depletion, whereas later reductions in Vcmax and Rubisco abundance 396 indicate selective protein retooling rather than wholesale N loss. Despite positional 397 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 18 differences in export capacity, apical and basal leaves presented similar photosynthetic 398 responses, underscoring the systemic coordination of source–sink interactions within 399 branches. Together with complementary physiological, metabolic, and proteomic evidence 400 (Vincent et al. , 2025; Chen et al., 2026 under review ), these findings support a model in 401 which mature citrus leaves function as regulated C and N conduits under strong sink demand, 402 prioritizing the establishment of new photosynthetic capacity over the maintenance of 403 existing assimilation. 404 405 Supplementary data 406 Fig. S1. Illustration of the research material. The top, orange- and blue-colored leaves 407 indicate the 14C-labeled apical and basal leaves, respectively. 408 Table S1. Growth characteristics of the experimental trees. 409 410 Author contributions 411 SBH, YW, and CV planned and designed the research. SBH, QM, and SL conducted the 412 experiments. CV, SBH, and QM processed the data. SBH performed the statistical analysis. 413 CV and SBH drafted the manuscript. 414 415 Funding 416 This study was supported by grants from the United States Department of Agriculture, 417 National Institute of Food and Agriculture, Agricultural and Food Research Initiative project 418 2021-67013-33795 for funding this research. 419 420 Conflict of interest 421 The authors declare that they have no competing financial interests. 422 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 19 423 Data availability 424 The data that support the findings of this study are available from the corresponding author 425 upon reasonable request. 426 427 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 20

428 Chapin III F, Shaver G, Kedrowski R. 1986. Environmental controls over carbon, nitrogen 429 and phosphorus fractions in Eriophorum vaginatum in Alaskan tussock tundra. The Journal 430 of Ecology, 167-195. 431 Crous KY, Campany C, Lopez R, Cano FJ, Ellsworth DS . 2021. Canopy position affects 432 photosynthesis and anatomy in mature Eucalyptus trees in elevated CO2. Tree Physiology 41, 433 206-222. 434 Diepenbrock LM, Dewdney MM, Vashisth T, Kanissery R . 2019. 2022–2023 Florida 435 Citrus Production Guide: Pesticides Registered for Use on Florida Citrus. EDIS. 436 Duursma RA . 2015. Plantecophys-an R package for analysing and modelling leaf gas 437 exchange data. PloS one 10, e0143346. 438 Frank A. 1981. Effect of leaf age and position on photosynthesis and stomatal conductance 439 of forage grasses1. Agronomy journal 73, 70-74. 440 Gonzalez/i1Real M, Baille A . 2000. Changes in leaf photosynthetic parameters with leaf 441 position and nitrogen content within a rose plant canopy ( Rosa hybrida ). Plant, Cell & 442 Environment 23, 351-363. 443 Henning R, Brown R, Ashley D . 1979. Effects of Leaf Position and Plant Age on 444 Photosynthesis and Translocation in Peanut I. Apparent Photosynthesis and 14C 445 Translocation. Peanut Science 6, 46-50. 446 Iglesias DJ, Lliso I, Tadeo FR, Talon M . 2002. Regulation of photosynthesis through 447 source: sink imbalance in citrus is mediated by carbohydrate content in leaves. Physiologia 448 Plantarum 116, 563-572. 449 Kitajima K, Mulkey SS, Samaniego M, Joseph Wright S. 2002. Decline of photosynthetic 450 capacity with leaf age and position in two tropical pioneer tree species. American Journal of 451 Botany 89, 1925-1932. 452 Kriedemann P . 1970. The distribution of 14C-labelled assimilates in mature lemon trees. 453 Australian Journal of Agricultural Research 21, 623-632. 454 Lamichhane JR, Alletto L . 2022. Ecosystem services of cover crops: a research roadmap. 455 Trends in plant science 27, 758-768. 456 Lemoine R, La Camera S, Atanassova R, Dédaldéchamp F, Allario T, Pourtau N, 457 Bonnemain J-L, Laloi M, Coutos-Thévenot P, Maurousset L, Faucher M, Girousse C, 458 Lemonnier P, Parrilla J, Durand M. 2013. Source-to-sink transport of sugar and regulation 459 by environmental factors. Frontiers in Plant Science 4. 460 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 21 Li S-y, Hussain SB, Vincent C. 2024. Response of carbon fixation, allocation, and growth to 461 source-sink manipulation by defoliation in vegetative citrus trees. Physiologia Plantarum 176, 462 e14304. 463 Li S-y, Vincent C . 2022. Root:shoot balance controls flush phenology and carbohydrate 464 translocation dynamics in citrus ( Citrus x sinensis) trunk. Physiologia Plantarum 174, 465 e13601. 466 Lima VF, Medeiros DB, Dos Anjos L, Gago J, Fernie AR, Daloso DM . 2018. Toward 467 multifaceted roles of sucrose in the regulation of stomatal movement. Plant Signaling & 468 Behavior 13, e1494468. 469 Liu H, Gao X, Fan W, Fu X . 2025. Optimizing carbon and nitrogen metabolism in plants: 470 From fundamental principles to practical applications. Journal of Integrative Plant Biology 471 67, 1447-1466. 472 Medeiros DB, Perez Souza L, Antunes WC, Araújo WL, Daloso DM, Fernie AR . 2018. 473 Sucrose breakdown within guard cells provides substrates for glycolysis and glutamine 474 biosynthesis during light-induced stomatal opening. The Plant Journal 94, 583-594. 475 Menezes J, Garcia S, Grandis A, Nascimento H, Domingues TF, Guedes AV, Aleixo I, 476 Camargo P, Campos J, Damasceno A. 2022. Changes in leaf functional traits with leaf age: 477 when do leaves decrease their photosynthetic capacity in Amazonian trees? Tree Physiology 478 42, 922-938. 479 Millard P, Grelet G-a. 2010. Nitrogen storage and remobilization by trees: ecophysiological 480 relevance in a changing world. Tree Physiology 30, 1083-1095. 481 Nebauer SG, Renau-Morata B, Guardiola JL, Molina R-V . 2011. Photosynthesis down-482 regulation precedes carbohydrate accumulation under sink limitation in Citrus. Tree 483 Physiology 31, 169-177. 484 Osorio S, Ruan Y-L, Fernie AR . 2014. An update on source-to-sink carbon partitioning in 485 tomato. Frontiers in Plant Science 5, 516. 486 Paul MJ, Foyer CH . 2001. Sink regulation of photosynthesis. Journal of Experimental 487 Botany 52, 1383-1400. 488 Poorter H, Niklas KJ, Reich PB, Oleksyn J, Poot P, Mommer L . 2012. Biomass 489 allocation to leaves, stems and roots: meta /i1 analyses of interspecific variation and 490 environmental control. New Phytologist 193, 30-50. 491 Proietti P, Palliotti A, Famiani F, Antognozzi E, Ferranti F, Andreutti R, Frenguelli G. 492 2000. Influence of leaf position, fruit and light availability on photosynthesis of two chestnut 493 genotypes. Scientia Horticulturae 85, 63-73. 494 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 22 Richardson AD, Keenan TF, Migliavacca M, Ryu Y, Sonnentag O, Toomey M . 2013. 495 Climate change, phenology, and phenological control of vegetation feedbacks to the climate 496 system. Agricultural and Forest Meteorology 169, 156-173. 497 Saathoff AJ, Welles J . 2021. Gas exchange measurements in the unsteady state. Plant Cell 498 Environ 44, 3509-3523. 499 Schloerke B, Cook D, Larmarange J, Briatte F, Marbach M, Thoen E, Elberg A, J C . 500 2024. GGally: Extension to 'ggplot2'. R package version 2.2.1. https://CRAN.R-501 project.org/package=GGally. 502 Smith MR, Rao IM, Merchant A . 2018. Source-sink relationships in crop plants and their 503 influence on yield development and nutritional quality. Frontiers in Plant Science 9, 1889. 504 Stinziano JR, McDermitt DK, Lynch DJ, Saathoff AJ, Morgan PB, Hanson DT. 2019. 505 The rapid A/Ci response. The New Phytologist 221, 625-627. 506 Syvertsen JP. 1985. CO 2 assimilation and water use efficiency of young expanding citrus 507 leaves. International Society for Horticultural Science (ISHS), Leuven, Belgium, 229-236. 508 Van Driesche RG, Carruthers RI, Center T, Hoddle MS, Hough-Goldstein J, Morin L, 509 Smith L, Wagner DL, Blossey B, Brancatini V, Casagrande R, Causton CE, Coetzee JA, 510 Cuda J, Ding J, Fowler SV, Frank JH, Fuester R, Goolsby J, Grodowitz M, Heard TA, 511 Hill MP, Hoffmann JH, Huber J, Julien M, Kairo MTK, Kenis M, Mason P, Medal J, 512 Messing R, Miller R, Moore A, Neuenschwander P, Newman R, Norambuena H, Palmer 513 WA, Pemberton R, Perez Panduro A, Pratt PD, Rayamajhi M, Salom S, Sands D, 514 Schooler S, Schwarzländer M, Sheppard A, Shaw R, Tipping PW, van Klinken RD . 515 2010. Classical biological control for the protection of natural ecosystems. Biological Control 516 54, S2-S33. 517 Vincent C, Hussain SB, Losada JM, Liesche J. 2025. Source–sink transport as a constraint 518 on photosynthesis and a driver of ecophysiological patterns. Plant Physiology 199, kiaf531. 519 Wallace A, Zidan ZI, Mueller RT, North CN . 1954. Translocation of nitrogen in citrus 520 trees. Proceedings of American Society of Horticultural Science 64, 87–104. 521 Werling BP, Dickson TL, Isaacs R, Gaines H, Gratton C, Gross KL, Liere H, 522 Malmstrom CM, Meehan TD, Ruan L, Robertson BA, Robertson GP, Schmidt TM, 523 Schrotenboer AC, Teal TK, Wilson JK, Landis DA . 2014. Perennial grasslands enhance 524 biodiversity and multiple ecosystem services in bioenergy landscapes. Proceedings of the 525 National Academy of Sciences 111, 1652-1657. 526 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 23 Xiao-Li W, Miao L, Chao-Su L, Mchugh ADJ, Ming L, Tao X, Yu-Bin L, Yong-Lu T . 527 2022. Source-sink relations and responses to sink–source manipulations during grain filling 528 in wheat. Journal of Integrative Agriculture 21, 1593-1605. 529 Xie S, Luo X . 2003. Effect of leaf position and age on anatomical structure, photosynthesis, 530 stomatal conductance and transpiration of Asian pear. Botanical Bulletin of Academia Sinica 531 44, 297-303. 532 Xiong H, Hu B, Wang J, Fu X-Z, Zhang Y, Shi X, Rennenberg H . 2025. Evergreen citrus 533 trees exhibit distinct seasonal nitrogen remobilization patterns between mature leaves and 534 bark. Horticulture Research 12, uhaf103. 535 Xiong H, Luo Y, Zhao H, Wang J, Hu B, Yan C, Yao T, Zhang Y, Shi X, Rennenberg H. 536 2024. Integrated proteome and physiological traits reveal interactive mechanisms of new leaf 537 growth and storage protein degradation with mature leaves of evergreen citrus trees. Tree 538 Physiol 44. 539 Zhao M, Liu Z, Hu Y, Yi S, Zhang Y, Hu B, Shi X, Rennenberg H. 2025. Carbon 540 metabolism and partitioning in citrus leaves is determined by hybrid, cultivar and leaf type. 541 Plant Physiology and Biochemistry 224, 109978. 542 543 544 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 24 Figure Legends: 545 Fig. 1. Comparison of photosynthetic parameters (Vcmax, J1400, TPU, Citrans1, and Citrans2) in the 546 apical and basal leaves of a branch at three phenological stages of sweet orange plants (Citrus 547 sinensis [L.] Osbeck). P indicates the effect of the leaf position across the branch, S indicates 548 the effect of the phenological stage, and PxS indicates the interaction between the leaf 549 position and the phenological stage. The error bars denote standard errors, and the points 550 denote means. 551 552 Fig. 2. Comparison of gas exchange parameters (E, A, Ci, gsw and gsw limitation) in the apical 553 and basal leaves of a branch at three phenological stages of sweet orange plants ( Citrus 554 sinensis [L.] Osbeck). P indicates the effect of the leaf position across the branch, S indicates 555 the effect of the phenological stage, and PxS indicates the interaction between the leaf 556 position and the phenological stage. The error bars denote the standard error, and the points 557 denote the means. 558 559 Fig. 3. Comparison of the total chlorophyll, nitrogen and Rubisco contents in the apical and 560 basal leaves of a branch at three phenological stages of sweet orange plants ( Citrus sinensis 561 [L.] Osbeck). P indicates the effect of the leaf position across the branch, S indicates the 562 effect of the phenological stage, and PxS indicates the interaction between the leaf position 563 and the phenological stage. The error bars denote standard errors, and the points denote 564 means. 565 566 Fig. 4. Correlation matrices among key physiological parameters in apical and basal citrus 567 leaves. The variables shown include the maximum rate of Rubisco carboxylase activity ( Vcmax), the 568 maximum rate of photosynthetic electron transport under saturating light ( J1400), net photosynthesis 569 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint 25 (A), stomatal conductance ( gsw), stomatal limitation ( gs-lim), and sucrose, starch, and Rubisco 570 contents. 571 572 Fig. 5. Stagewise correlation matrices among selected physiological traits in citrus leaves. 573 The variables shown include the maximum rate of Rubisco carboxylase activity ( Vcmax), the 574 maximum rate of photosynthetic electron transport under saturating light ( J1400), net photosynthesis 575 (A), stomatal conductance ( gsw), stomatal limitation ( gs-lim), and sucrose, starch, and Rubisco 576 contents. 577 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted March 11, 2026. ; https://doi.org/10.64898/2026.03.09.710566doi: bioRxiv preprint