Source: UNIVERSITY OF FLORIDA submitted to NRP
WIDENING THE BOTTLENECK:SOURCE-SINK PARTITIONING & TRANSLOCATION FOR OPTIMAL PHOTOSYNTHESIS IN PERENNIAL FRUIT CROPS SOURCE-SINK RELATIONS, CARBOHYDRATE TRANSLOCATION, PHOTOSYNTHESIS, NET ASSIMILATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1025303
Grant No.
2021-67013-33795
Cumulative Award Amt.
$495,000.00
Proposal No.
2020-03614
Multistate No.
(N/A)
Project Start Date
Jan 15, 2021
Project End Date
Jul 14, 2025
Grant Year
2021
Program Code
[A1152]- Physiology of Agricultural Plants
Recipient Organization
UNIVERSITY OF FLORIDA
G022 MCCARTY HALL
GAINESVILLE,FL 32611
Performing Department
(N/A)
Non Technical Summary
Source-sink attenuation of photosynthesis reduces crop net assimilation rate (NAR). To address this reduction, our goal is to quantify the relationships between allocation, translocation and photosynthesis in woody perennial crops and to develop strategies to optimize these relationships. Our model is Citrus because it has a diverse multi-species germplasm and a large industry. The purpose of this proposal is to quantify the interaction of source-sink allocation with phloem transport speeds and photosynthesis in Citrus, both on individual and population levels. Our central hypothesis is that selection for greater relative sink allocation will lead to greater NAR until phloem loading and transport become limiting.Our specific objectives are to:1.Quantify the relative control of source export, sink import and phloem translocation on photoassimilate flux in Citrus. We will combine growth analysis with a unique approach to measuring 14-C translocation rates in vivo to quantify source-sink and translocation fluxes.2.Determine the effect of source export on regulation of photosynthesis. Rapid A/Ci response methods will characterize leaf-level photosynthetic activity.3.Apply the relationships described in Objectives 1 and 2 on a population level in breeding families with observed variations in source-sink relationships. The same methods will be employed to assess source-sink optimization of NAR in a breeding population, including assessing source-sink transport to fruits.This project will address the program priority of "improving plant performance and productivity" by providing breeders with selectable traits to enhance NAR of cultivated citrus by quantifying the relationship between source-sink allocation, translocation and photosynthesis.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20609991020100%
Knowledge Area
206 - Basic Plant Biology;

Subject Of Investigation
0999 - Citrus, general/other;

Field Of Science
1020 - Physiology;
Goals / Objectives
1. Quantify the relative control of source export, sink import and phloem translocation onphotoassimilate flux in Citrus. We expect that increasing sink:source ratio will lead to higher leafexport rates and translocation speeds until either transport capacity or loading capacity becomelimiting.2. Determine the effect of source export on regulation of photosynthesis. We expectphotosynthetic activity will increase with increasing sink partitioning and will be limited at thesame sink:source as leaf export.3. Apply the relationships described in Objectives 1 and 2 on a population level in breedingfamilies with observed variations in source-sink relationships. We expect a breeding family withdiverse source-sink allocation characteristics to demonstrate the same relationship betweensink:source and net assimilation as on an individual level.
Project Methods
Objective 1Hypothesis 1.1: Tetraploidy will increase partitioning to roots. This hypothesis will be useful intesting Hypothesis 1.4, because we will exclude potential genetic variation.Hypothesis 1.2: Sink unloading is the dominant process determining carbohydrate translocationrate in the short term.Hypothesis 1.3: Increasing relative sink allocation increases translocation speed and leaf exportrate until transport or loading becomes limiting.Hypothesis 1.4: Source loading is controlled by sink unloading in the long term.Objective 1 MethodsExperiment 1.1For this experiment we will use diploids and autotetraploids of:1. 'Valencia' (C. sinensis), source preferring (Rodríguez-Gamir et al., 2010).2. 'W. Murcott' (Tangor type, C. reticulata), sink preferring (Li et al., 2003).3. Cybrids of Mandarin #304 (C. reticulata) with 'G1' satsuma (C. unshiu or C. reticulata)cytoplasm. Growth habit not observed.4. Cybrids of Volkamer lemon (C. volkameriana) with C. amblycarpa cytoplasm. Growthhabit not observed.?At approximately 6-months after rooting, plants will be between 30 and 60 cm in height. Ten plantswill be sampled for destructive measurement at 6-months for destructive measures, and another 10at the second sampling date. Destructive samples for growth analysis:1. Dry weights (DW) of roots, stem and leaves.2. Leaf area (LA) and number.Experiment 1.2To test Hypotheses 1.2, 2 varieties of divergent source-sink patterns, 'Cleopatra' mandarin (C.reticulata) and US-942 (C. reticulata x P. trifoliata) will be purchased from a local nursery. IfHypothesis 1.1 is confirmed, this experiment will be repeated on the most divergent diploidtetraploidpair. Plant maintenance and experimental design will be the same as Experiment 1.1,except that this experiment will have 4 replications, which we have found to be sufficient fordetection of treatment differences (Vincent, unpublished data).Translocation will be measured twice in each plant. The first labeling and measurement will beprior to any treatment. Treatments, as depicted in Figure 5, will be:1. Girdling: A cold-block girdle will be induced in the stem above the 1st stem detector afterthe pulse passes that detector.2. Pruning approximately 1/3 of the root mass prior to loading.3. Pruning approximately half of the root mass prior to loading, and girdling after the pulsepasses the first stem detector.Measurements14C labeling and detectionEstimation of sink strengthExperiment 1.3To quantify the response curve of translocation and foliar export to sink:source ratio (Hypothesis1.3), we will use the same pairs of varieties as Experiment 1.2. In this experiment we will test theeffects of various levels of partial defoliation. Plants will be maintained as in Experiment 1.1. Sixtreatments will be applied:1. 0% defoliation2. 20% defoliation3. 40% defolation4. 60% defolation5. 80% defoliation6. Removal of all but 3 leaves.Treatments will allow the full range of potential translocation responses within the variety. Designwill be arranged as a randomized complete block, but trees will be removed from the greenhousefor measurement. Newly emerging shoots will be removed to maintain a constant leaf area, as inSyvertsen (1994). Leaf area and dry weight of the removed leaves will be measured. Translocationand leaf export will be measured as in Experiment 1.2 before defoliation and 1-week afterdefoliation. After measuring translocation, plants will be harvested destructively to measure leafarea and stem, leaf and root dry weights.Experiment 1.4To test Hypothesis 1.4, genotypes with divergent source:sink will be reciprocally grafted. IfExperiment 1.1 confirms Hypothesis 1.1, we will use diploid-autotetraploid pairs to reduce geneticvariability. We will graft 3 pairs including self-grafting, for a total of 4 combinations per pair (A/B,B/A, B/B, A/A). Each combination will be replicated 10 times, for a total of 120 plants.Propagation, plant care, and experimental design will be as in Experiment 1.1. Translocation willbe measured on a subset of 4 plants per combination, while growth analysis from destructivemeasurements will be performed on all 10 replicates per combination. In this experiment, the same14C translocation, photosynthetic measurements, and growth analysis will be performed as inExperiment 1.1. Thus, the relative control of source and sink over time on growth and on allocationcan be quantified.Objective 2Hypothesis 2.1: A and Vcmax co-vary with leaf export rate.Hypothesis 2.2: Leaf respiration and triose phosphate use limitation are negatively correlated withleaf export rate.Objective 2 MethodsHypotheses 2.1 and 2.2 will be tested using Experiments 1.3 and 1.4. Experiment 1.3, using partialdefoliation, will allow us to test the effect of short-term source-sink modifications within genotypeon photosynthesis. Experiment 1.4 will allow us to test the effects of longer-term allocation effectsvia grafting.Leaf RespirationRapid A/Ci curvesObjective 3?Hypothesis 3.1: The source-sink allocation relationships described by Experiments 1.1-1.4 operateon a population scale.Hypothesis 3.2: The source-sink allocation relationships described by Experiments 1.1-1.4contribute to fruitlet sink demand.Objective 3 MethodsExperiment 3.1Three breeding populations will be assessed for source-sink allocation and photosynthesis. Thesefamilies are the progeny of crosses between:1. Sweet orange (C. sinensis) x Poncirus trifoliata (Citrange; >100 individual F1 hybrids areavailable)2. Other P. trifoliata crosses with:a. Sour orange (C. x aurantium) (>10 individuals)b. Grapefruit (C. x paradisi) (>10 individuals)c. Kinkoji (C. obovoidea) (>10 individuals)3. C. ichangensis(ichang) crosses with:a. Palestine sweet lime (PSL, C. limettiodes) (>60 individual hybrids available);b. Cleopatra mandarin (C. reticulata) (>15 individuals)c. Swingle citrumelo (C. x paradisi x P. trifoliata) (>15 individuals)Experiment 3.2Fifty genotypes of divergent source-sink characteristics as determined in Experiment 3.1 will bepropagated by cuttings as in Experiment 1.1. At least 10 genotypes will be selected for each of theabove-mentioned breeding families. Six plants per genotype will be propagated to allow for 3individuals in each of 2 destructive measures. Measurements will be the same as in Experiment1.1. Thus, we will be able to test the relationship between characteristics across the population,and to test the hypotheses on a population basis. Depending on the results of these experimentsand those of Experiment 1.1, each quantitative trait will be mapped in the citrange and PSL-ichangfamilies using routine QTL mapping approaches to identify genomic regions associated withspecific traits. Depending on the magnitude and size of QTL intervals identified, bioinformaticinvestigations can be conducted with existing and new citrus reference genomeshttps://www.citrusgenomedb.org/species/sinensis/genome1.0identify specific genes that may be involved with the key physiological processes that underliewhole plant carbon dynamics.Experiment 3.3To test Hypothesis 3.2, we will implement an experiment similar to Experiments 1.3 and 1.4,except that it will isolate small fruitlets as the sink of interest. If more than one fruitlet ispresent, the additional fruitlets will be removed. Leaves will be removed selectively to producefour treatment groups:1. 100% control with no girdling2. 100%: girdling with 7-8 leaves remaining (none removed)3. 50%: girdling with 4 leaves remaining (3-4 removed)4. 25%: girdling with 2 leaves remaining (5-6 removed)Four shoots of each treatment group will be produced.?

Progress 01/15/24 to 01/14/25

Outputs
Target Audience:Our target audience is the scientific community and bland biologists specifically.In the past year the PhD student on this project has delivered several professional presentations or posters of her work, and the PI has also delivered one presentation. One peer-reviewed publication has resulted and two more are in process. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Professional presentations, and also 2 perr-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals?We will complete analysis of the existing experiments and apublish additional work summing total fluxes.

Impacts
What was accomplished under these goals? 1. We have demonstrated that transport speeds do not appear to be affected by source-sink variation. Additionally stem reserves appear to have a major role in buffering these fluxes, though in the medium term source-sink increases increase leaf export. 2. Decreasing source:sink reduces fluxes but does not greatly affect photosynthetic rates, while increasing source-sink demand only minimally affects fluxes, but has a strong effect on photosynthesis (Li et al. 2025). 3. Analysis is in progress on completed experiments.

Publications

  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Li, S. Y., Hussain, S. B., & Vincent, C. (2024). Response of carbon fixation, allocation, and growth to source?sink manipulation by defoliation in vegetative citrus trees. Physiologia Plantarum, 176(3), e14304.
  • Type: Peer Reviewed Journal Articles Status: Published Year Published: 2024 Citation: Hussain, S. B., Stinziano, J., Pierre, M. O., & Vincent, C. (2024). Accurate photosynthetic parameter estimation at low stomatal conductance: effects of cuticular conductance and instrumental noise. Photosynthesis Research, 160(2), 111-124.


Progress 01/15/23 to 01/14/24

Outputs
Target Audience: Nothing Reported Changes/Problems:No technical problems have arisen. Results were surprising and follow-up is underway. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?Journal articles and conference presentations. What do you plan to do during the next reporting period to accomplish the goals?We are now focusing on analyses of completed experiements.

Impacts
What was accomplished under these goals? 1 and 2. We found that increasing sink:source did increase Vcmx, Jmax, and TPU, but did not find any corresponding changes in stem transport or leaf export. We are engaged in follow-up experiments to understand this phenomenon more effectively. 3. Experiments complete. Analysis in progress.

Publications

  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Li, S. Y., Hussain, S. B., & Vincent, C. (2024). Response of carbon fixation, allocation, and growth to source?sink manipulation by defoliation in vegetative citrus trees. Physiologia Plantarum, 176(3), e14304.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Hussain, S. B., Stinziano, J., Pierre, M. O., & Vincent, C. (2024). Accurate photosynthetic parameter estimation at low stomatal conductance: effects of cuticular conductance and instrumental noise. Photosynthesis Research, 1-14.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: D. Alade and C. Vincent. 2024. Impact of Huanglongbing (HLB) on Source-Sink Dynamics and Photosynthesis in Citrus Plants. VII International Research Conference on Huanglongbing. Riverside, California. March 25, 2024.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: C. Vincent, D. Alade, J. Robledo, M. Keeley, S.B. Hussain, and A. Levy. 2024. A whole-plant physiological framework for HLB. VII International Research Conference on Huanglongbing. Riverside, California. March 25, 2024.


Progress 01/15/22 to 01/14/23

Outputs
Target Audience:The target audience of this project is crop breeders, plant physiologists, and agricultural scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate student and post-doctoral scholar have been trained in radioisotope usage, radiation safety, and gas exchange approaches. How have the results been disseminated to communities of interest?Results were presented to the international Plant Vascular Biology conference in Berlin Germany, July 2022. What do you plan to do during the next reporting period to accomplish the goals?We will complete the phenotypic analysis and the diploid-tetraploid experiments.

Impacts
What was accomplished under these goals? Objectives 1 and 2. Experiments assessing the impact of increasing source:sink gradients brought surprising results. We found no increase in the leaf export rate nor in the transport speed, while the maximum rate of carboxylation (Vcmax), of electron transport (Jmax), and triose phosphate utilization. Meanwhile results suggested that the position of the leaf on the stem could impact export rate. Rather than an increase in gas C fixation parameters with moderate degrees of defoliation with diminishing returns at higher defoliation, we observed stronger increases with increasing defoliation up to our maximum (leaving only 1 leaf per branch tip). Defoliation induced new shoot growth.We followed up with experiments to assess the impact of new shoot growth ("flushing") and leaf position on carbon fixation and export. We found, as previously, that flushing increased C export in a position dependent manner: leaves close to the apex with new (sink) shoots had the highest C export rates. However, carbon fixation variables decreased as C export increased. We are now assessing the impact of flushing on N dynamics to compare with the carbon dynamics. Thus far our conclusions are: 1, The type of sink as well as its magnitude impact C fixation dynamics. Roots require C from sources, but acquire N directly, and higher root sink demand relative to leaf area leads to upregulated C fixation. Shoots require both N and C and therefore may be competing with existing sinks for N for rubisco. 2. C transport speed is stable in citrus. Short-term source-sink changes do not appear to impact transport speed. 3. Sink proximity impacts leaf C export. 4. Both conclusions 2 and 3 lead us to suspect an important role of stem nonstructural carbohydrates(NSCs) in buffering supply and demand. We are currently implementing experiments to address C:N tradeoffs and the storage of stem NSCs. Additionally, we have prepared populations of diploid-autotetraploid pairs to assess these dynamics in differing but genetically identical individuals. Objective 3. Several different types were crossed in Spring of 2022. Crosses included crosses of among mandarin (C. reticulata), C. ichangensis, grapefruit (C. x paradisi), pumelo (C. maxima), and Poncirus trifoliata x Citrus hybrids. The resulting seedswere planted and grown for at least 6 months. The destructive analysis of the resulting 415 indifviduals was implemented in December 2022, and we are now sifting through the data. Variables include organ (leaf, stem, root) dry weights, A/Ci curves on 50 select individuals, scanning of root systems, plant heigth, canopy branching patterns, leaf morphology, and chlorophyll fluorescences.Samples were gathered to measur foliar carbohydrates, N, and Chlorophyll content.After phenotypic analysis genome wide associative selection will be performed.

Publications

  • Type: Journal Articles Status: Other Year Published: 2022 Citation: Li, S.Y., S.B. Hussain, C.I. Vincent. 2023. LOW SOURCE:SINK REGULATES LEAF PHOTOSYNTHESIS AND NEW GRWOTH BUT LEAF EXPORT WAS UNAFFECTED IN CITRUS. J. Plant Physiol. (in preparation).
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: C. Vincent, S.-y. Li, S.B. Hussain, M. Keeley, and A. Levy. 2022. Invited oral presentation. Leaf carbon fixation, export, and transport in citrus trees exhibit highly buffered responses to source-sink dynamics. Plant Vascular Biology 2022. Berlin, Germany. July 21, 2022.


Progress 01/15/21 to 01/14/22

Outputs
Target Audience: Nothing Reported Changes/Problems:There are no major changes. However, we have been dramatically slowed in starting the student and post-doc by immigration processes. The postdoc began in September and the PhD student began in January 2022, each approximately 6 months later than anticipated. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?In the next year we will continue advancing on Objectives 1 and 2. We will begin making progress on Objective 3.

Impacts
What was accomplished under these goals? 1. We have tested the hypothesis that increasing source:sink ratio will lead to higher export rates and translocation speeds, with some surprising preliminary results: Transport speed did not change across a source:sink gradient. Leaf export rate did not change according to source:sink gradient. This actually appeared to respond to leaf position in the canopy. Maximum rate of carboxylation did increase with source:sink gradient. Theseresults are surprising and must be confirmed, but this challenges the conventional understanding of the relationship between export processes and the regulation of carbon fixation. Follow-up experiments are in process now. 2. See 1. 3. Plant populations are being developed.

Publications

  • Type: Journal Articles Status: Submitted Year Published: 2022 Citation: Christopher Vincent, Myrtho O Pierre &, Bilal Hussain P, and Joseph Stinziano p. 2021. Racing against stomatal attenuation: rapid CO2 response curves more reliably estimate photosynthetic capacity than steady state curves in a low conductance species. New Phytologist. https://www.biorxiv.org/content/10.1101/2020.08.28.270785v1.full.pdf