Monitoring of the phenological stage of Lactuca sativa cv. Rex plants using the Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie (BBCH) scale. (A) Description of the principal growth stage (PGS) and secondary growth stage (SGS) as defined by the BBCH scale. (B) Overhead image of plants used to determine head area with ImageJ software. (C) Progression of newly unfolded leaves from 14 to 30 d after sowing (DAS) in response to the application of Sugar Mover® Premier or the untreated control. (D) Proportional head area over time in response to the application of Sugar Mover® Premier or the untreated control expressed as a percentage of the final area (504 cm2) at harvest for the untreated control. The horizontal dotted line (- - -) marks the point when plants reach 90% of their final area, representing typical size, shape, firmness, and SGS 9 on the BBCH scale. Vertical dotted lines indicate the time (DAS) when each treatment reached SGS 9. Asterisks denote statistical differences according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis. ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
(A) Soil plant analysis development (SPAD) index, (B) shoot length (SL), (C) root length (RL), (D) shoot dry matter (SDM), (E) root dry matter (RDM), and (F) total dry matter (TDM) of Lactuca sativa cv. Rex plants in response to the application of Sugar Mover® Premier or untreated control. Statistical differences were determined according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis, with P ≤ 0.05 indicating significance (n = 24). ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. DAT = days after transplanting to deep-water culture systems.
Number of leaves and corresponding leaf area (cm2) of Lactuca sativa cv. Rex plants in response to the application of Sugar Mover® Premier or the untreated control at 7 DAT (A), 14 DAT (B), 21 DAT (C), 28 DAT (D), and 35 DAT (E). The letter “L” denotes the leaf number. The darker dashed line at the top of each graph indicates the maximum leaf area achieved by the control treatment at each sampling point set at 100% leaf expansion. The two lighter dashed lines represent 60% and 30% leaf expansion, respectively. Leaves with more than 60% leaf expansion were classified as source leaves, whereas those with less than 30% were categorized as sink leaves. Leaves exhibiting 30% to 60% leaf expansion were designated as transition leaves. Categorization based on leaf expansion was performed as described by Turgeon (1989) and Fellows and Geiger (1974). DAT = days after transplanting to deep-water culture systems.
Foliar parameters of Lactuca sativa cv. Rex leaves in response to the application of Sugar Mover® Premier or the untreated control. (A) Frequency of leaves categorized as sinks, sources, or in transition between these stages in untreated control plants (left bar) and treated plants (right bar). (B) Leaf length (LL; cm), (C) leaf width (LW; cm), and (D) leaf area (LA; cm2). Statistical differences were determined according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis, with P ≤ 0.05 indicating significance (n = 24). ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. DAT = days after transplanting to deep-water culture systems.
Growth indices calculated based on the growth and dry matter parameters of Lactuca sativa cv. Rex plants. (A) Relative growth rate (RGR; increase in dry mass per unit plant dry mass and time; g·d−1). (B) Net assimilation rate (NAR; dry matter gain per unit leaf area per unit time; g·m−2·d−1). (C) Leaf area ratio (LAR; leaf area per unit total plant biomass; m2·g−1). (D) Leaf mass ratio (LMR; the ratio of leaf mass to plant mass; g leaf/g plant). (E) Specific leaf area (SLA; leaf area per unit leaf dry mass; m2·g−1). (F) Root-to-shoot ratio (RSR). Each boxplot represents the average of 12 values (n = 12). Statistical differences were determined according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis, with P ≤ 0.05 indicating significance. ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. DAT = days after transplanting to deep-water culture systems. Sampling points = 7, 14, 21, 28, and 35 d after treatment impositions.
Proportional variation in the contents of protein, amino acids, reducing sugars (RS), total soluble sugars (TSS), sucrose, and starch in the roots, outer leaves, and head leaves of Lactuca sativa cv. Rex across treatments at each sampling point. Within each sampling point, the left bar represents the control treatment, and the right bar represents the Sugar Mover® Premier treatment. DAT, days after transplanting to deep-water culture systems. T1 = control; T2 = application of the cytokinin B-Mo-based product. For detailed values and statistical analysis of each macromolecule, refer to Table 1.
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An understanding of how amendments influence the sink-to-source relationship in leafy crops can be used to optimize plant resource allocation for enhanced growth and quality. Variations in growth rates and carbon pools across individual leaves or groups of leaves at similar developmental stages allow us to comprehend plant strategies of carbon allocation and partitioning. We hypothesized that products enhancing the carbon source-to-sink relationship during leaf development can increase growth and dry matter accumulation. This project aimed to determine whether exogenous applications of a cytokinin-B-Mo-based product during the leaf development of lettuce plants impact the carbon source-to-sink relationship, thus influencing plant growth and quality. The experiment was a complete randomized design with two treatments: a negative control and the application of the product twice during the growing cycle. Each experimental unit consisted of a deep-water culture reservoir with three lettuce plants. Destructive sampling was conducted five times throughout the cycle. At each sampling time (n = 4 per experimental run), the phenological stage was determined, and measurements of root and shoot length, root and shoot dry matter, leaf length, leaf width, leaf area, chlorophyll contents, and nonstructural carbon contents were performed. These data were used to estimate growth indices. The results indicated that the cytokinin-B-Mo-based product increased the number of true unfolded leaves by 1 ± 0.4 and the overall size of the lettuce head by 9%. The treated lettuce reached marketable size 4 days earlier than that of the control treatment. Statistically significant differences were observed in shoot and root dry matter accumulation and foliar length and width at some sampling points. Some growth indices indicate an increase in leaf surface area investment and enhanced conversion efficiency of assimilates into biomass in plants treated with the product. Plants exhibiting these alterations had higher sucrose and total soluble sugar contents. There was a noticeable pattern of higher concentrations of nonstructural carbohydrates, proteins, and amino acids in the leaves compared with those in the roots across all plants and treatments. Overall, the cytokinin-B-Mo-based product appears to strengthen the source-to-sink relationship during lettuce development, resulting in a high-quality plant within a shorter timeframe.
Crop yield is highly regulated by the ability of the plant to produce, reallocate, and use carbohydrates. Therefore, the source-to-sink relationship plays a pivotal role in determining plant growth rates by governing the allocation of resources within the plant (Aye et al. 2020; Rossi et al. 2015). Unraveling the signaling network governing the carbon (C)/nitrogen (N) status and the tradeoffs in carbohydrate allocation across organs and processes is important to effectively channeling available resources where they are most needed for growth and development (Paul et al. 2020; Rossi et al. 2015) and, consequently, yield. Leaves undergo two developmental transitions during plant development, the sink-to-source transition and the mature-to-senescent transition (Côrrea 2008), which are particularly important in the context of leafy crop production (e.g., lettuce). Leaves embody within the same organ the metabolic changes associated with sinks and sources. Therefore, an understanding of the developmental transitions and C allocation in leaves provides valuable insights into optimizing resource allocation strategies to enhance plant growth, crop yield, and quality.
The sink-to-source transition in leaves involves a shift in the distribution and accumulation of resources to match metabolic requirements and structural and anatomical changes throughout the leaf ontogeny (Carvalho et al. 2016). Young expanding leaves (sinks) are net C importers with low photosynthesis rates, high respiration rates, and highly activated biosynthesis pathways (Côrrea 2008; Meng et al. 2001). As the leaves expand and mature, their sink strength decreases and the rate of photosynthesis gradually increases, leading to a decrease in their dependence on the import of carbohydrates. Then, the leaves start exporting metabolites from their photosynthetic assimilation, thus becoming net exporters (sources) (Turgeon 1989). The transition of the mesophyll from sink to a fully developed source tissue occurs in a basipetal direction over several days. This changing ratio of imported to photosynthesized incoming C indicates that the leaf ontogeny plays a critical role in shaping the temporal and spatial patterns of C distribution and partition. In some cases, the C metabolism of single leaves within the same rosette differs by developmental stage, thus highlighting the importance of understanding the C pool and the physiological insights of individual leaves or groups of leaves at similar developmental stages rather than the total pool of the rosette (Dethloff et al. 2017). Therefore, an understanding of the carbohydrate pool of leaves in different developmental stages is crucial to comprehending how the plant modulates and distributes the available C resources.
The sink-to-source relationship can be approached from the source metabolism perspective, which is primarily centered on optimizing photosynthesis. Various strategies target enhancing rubisco activity, different photosynthetic metabolisms (C3, C4, and CAM), and C-concentrating mechanisms, among others (Carmo-Silva et al. 2015; Ort et al. 2015; Rosado-Souza et al. 2023). Alternatively, enhancing sink strength efforts have been directed toward augmenting sink capacity by overexpressing or increasing the activity of metabolite transporters or enzymes like sucrose synthase (Ludewig and Sonnewald 2016). The transport of the assimilated C, including long-distance transport from source leaves and sugar movement systems for phloem loading, plays a pivotal role in modulating allocation to desired sinks and determining the overall biomass allocated to harvestable organs (Fernie et al. 2020; Ludewig and Sonnewald 2016; Rossi et al. 2015). Our research explored the sink and transport sides of the carbohydrate network using a cytokinin B-Mo-based product that aims to strengthen the plant’s source-to-sink relationship by shifting the movement of sugars toward the sink organs. By assessing the allocation and utilization of assimilated C by sink organs through measurements of growth rates, biomass accumulation, and nonstructural carbohydrates (NSCs), it is possible to elucidate whether this product influences the transition between sink and source phases in the leaves. Furthermore, NSCs play an essential role in indicating a plant’s C status, and measuring the NSC stock’s size and fluctuations during periods of varying demand is a quick and straightforward approach to directly assessing any shortage or surplus affecting the balance of the source–sink activity (Körner 2003).
Cytokinins are a group of adenine derivatives biosynthesized in roots and transported via the xylem to shoots and leaves and key regulators of numerous processes in plant development and growth (Mok et al. 2000). The analysis of cytokinin-deficient plants has shown that cytokinins regulate several parameters that determine the source or sink strength of tissues, such as C fixation, assimilation, and partitioning of primary metabolites (Roitsch and Ehness 2000; Werner et al. 2008). Cytokinins have been positively correlated with the expression of various transcripts, proteins, and photosynthesis-related genes of the chloroplast genome involved in photosynthetic reactions (Brenner et al. 2005). Furthermore, cytokinins have been shown to upregulate the expression of genes encoding cell wall invertases, thus impacting sucrose cleavage (Ehness and Roitsch 1997; Roitsch and Ehness 2000) and sink tissue cell proliferation and differentiation (Oshchepkov et al. 2020), which are crucial for sink strength.
Boron (B) is an essential micronutrient for plants (Warington 1923) that is necessary for the growth of meristematic tissues, cell division, membrane function, and assimilate partitioning (Pereira et al. 2021; Rehman et al. 2018; Shireen et al. 2018). Under B-deficient conditions, the plant phloem transport is affected, thus reducing sink capacity, which alters assimilate translocation and distribution to growing regions (Marschner 1995; Rehman et al. 2018; Wimmer and Eichert 2013). Molybdenum (Mo) is a trace element required for plant growth that is predominantly found as an integral part of an organic pterin complex called the Mo cofactor. The Mo cofactor binds Mo-requiring enzymes, such as N assimilation and N-fixing enzymes (Kaiser et al. 2005; Rana et al. 2020). Plants incapable of using Mo cofactor are shown to be defective in nitrate reduction (Rana et al. 2020), leading to inhibited nitrate assimilation activity. This could potentially affect the C/N homeostasis associated with the regulation of the source-to-sink relationship. Furthermore, research suggests that Mo deficiencies can also impact the expression of genes involved in metabolism, transport, development, and signal transduction (Ide et al. 2011).
We hypothesize that exogenous application of a cytokinin and B-Mo-based product during the plant development of leafy crops can influence plant growth and quality, potentially affecting sugar allocation between sources and sinks. In this project, we tracked the growth, dry matter (DM) accumulation, and partitioning of NSCs when Sugar Mover® Premier (Stoller USA, Houston, TX, USA), a product containing cytokinin, B, and Mo, was applied to hydroponic lettuce plants (Lactuca sativa cv. Rex).
Lactuca sativa Rex seeds (Johnny’s Selected Seeds, Winslow, ME, USA) were sown in 1-inch2 oasis cubes (OASIS Grower Solutions, Kent, OH, USA) placed in black propagation trays (11 × 21 inch). The germination process and seedling development took place in a growth chamber set at 25 °C (80 °F), humidity of 60%, and a 12-h/12-h day/night cycle under fluorescent and incandescent lamps (14.6 mol·m−2·d−1). At 14 d after sowing (DAS), the lettuce seedlings were transplanted into deep-water culture (DWC) systems. Each system consisted of a 1-inch-thick extruded polystyrene foam (Styrofoam™; DuPont de Nemours, Inc., Wilmington, DE, USA) with 15- × 19-inch dimensions on top of a 7 1/8-gal DWC reservoir (Rubbermaid, Atlanta, GA, USA) with a 4- × 2- × 2-inch air stone (Vivosun, ON, Canada) connected to an aerator (General Hydroponics, Santa Rosa, CA, USA) and a digital display aquarium heater (Hygger, Shenzhen City, Guangdong Province, China) set at 21 °C (70 °F). Each polystyrene foam had three holes in a triangle arrangement with a net basket containing a seedling.
The DWC systems were placed on benches in a glass greenhouse with a heating set point of 20 °C and a ventilation set point of 25 °C. The natural photoperiod was supplemented with high-pressure sodium lamps (C1000S52; Agrolite XL, Philips, Amsterdam, Netherlands) operating from 7:00 AM to 7:00 PM during the following two experimental runs: Oct to Dec 2022 and Jan to Mar 2023. Throughout the experiment, the average photosynthetic photon flux density under the high-pressure sodium fixtures was 101.33 μmol·m−2·s−1. Each DWC reservoir had 24 L of nutrient solution containing 19N–8P–13K at 78.8 mg·L−1 N (JR Peters Inc., Allentown, PA, USA). The pH of the nutrient solution was maintained between 5.8 and 6.2, and electrical conductivity was approximately 1.5 mS/cm for early vegetative stages (14–30 DAS) or 2.0 mS/cm for later production (31–50 DAS).
Sugar Mover® Premier (hereafter referred to as cytokinin B-Mo-based product) is an EPA-registered product containing cytokinin (0.003% as kinetin), B (8%), and Mo (0.004%). The label dosage recommended for spinach and lettuce is 1.2 to 2.4 L·ha−1. Because there is no specified rate for hydroponic systems, the application rate was extrapolated from the manufacturer’s instructions for root dip and watering solution used during transplanting. The product was applied directly into the nutrient solution at a rate of 0.29% (v/v) twice: 14 DAS (when the plants were transferred from the growth chamber to the DWC systems) and 35 DAS [or 21 d after transplant (DAT)]. The final concentration of the active ingredient was 0.001 mg·L−1 cytokinin, 2.4 mg·L−1 Bo, and 0.001 mg·L−1 Mo.
The experiment was a completely randomized design with two treatments consisting of a negative control [treatment 1 (T1)] and the application of cytokinin B-Mo-based product twice during the growing cycle [treatment 2 (T2)]. The experimental unit consisted of a DWC reservoir with three lettuce plants, with one experimental unit per repetition and 20 repetitions per treatment. Destructive sampling was conducted 7, 14, 21, 28, and 35 DAT to DWC systems. At each sampling point, four DWC reservoirs were randomly selected and destructively sampled per treatment (n = 4), and the experiment was run twice (n = 8). Because both experimental runs were analyzed together, 24 plants (8 replicates × 3 plants per experimental unit) per treatment were measured at each sampling point. After destructive sampling, the shoot (outer and head leaves) and root DM from 24 plants per treatment were homogenized, ground, and sieved separately to assess nonstructural carbohydrate partitioning. For each organ—roots and outer leaves at 7, 14, and 21 DAT and roots, outer leaves, and head leaves at 28 and 35 DAT—four technical replicates of 0.2 g were taken from the homogenized material per experimental run, resulting in a total of eight samples per organ, per treatment, analyzed at each sampling point.
To evaluate whether the application of the cytokinin B-Mo-based product had any effect on the development of the plant and the duration of the growing cycle, we assessed the change in the development phases over time through the Biologische Bundesanstalt, Bundessortenamt, und Chemische Industrie (BBCH) scale (Feller et al. 1995; Meier et al. 2009; Taghavi et al. 2022). For vegetables forming heads as lettuce, the scale standardizes a coding for nine phenological growth stages. We followed principal growth stage (PGS) 1 and PGS 4 with their respective secondary growth stages (SGS) (Fig. 1A); PGS 1 characterizes leaf development (main shoot) through the development of true unfolded leaves over time. From 14 DAS (0 DAT), every other day, we followed the progression of newly unfolded leaves, starting with the chronologically youngest leaf in the center and progressing to the following successively older leaves (following a circular pattern clockwise). Additionally, PGS 4 characterizes the development of the harvestable vegetative plant parts considering the percentage of the final expected head size increase over time. Therefore, at 30 DAS (16 DAT), when we visualized the formation of a rosette arrangement that characterizes the beginning of the head formation process, we ceased counting the number of leaves and instead measured the area/size of the head. To obtain accurate measurements, we captured an overhead photograph of the plants (Fig. 1B), which was subsequently analyzed using Image J software version 1.53 (National Institutes of Health, Bethesda, MD, USA) to estimate the area.
Citation: HortScience 60, 2; 10.21273/HORTSCI18281-24
We evaluated whether the application of the cytokinin B-Mo-based product had any effect on shoot and root development. At all sampling points, the plants were separated between the root and shoot. The biomass and leaf chlorophyll content were measured from shoots. The leaf chlorophyll content in five fully expanded leaves from each plant was indirectly estimated using a handheld chlorophyll meter (SPAD 502; Spectrum Technologies, Inc., Aurora, IL, USA). The root length and biomass were measured. The dry weights were obtained by putting the shoot and root in paper bags inside an oven at 72 °C for 48 h. All weights were determined using a calibrated digital analytical balance.
We evaluated whether the application of the cytokinin B-Mo-based product had any effect on certain foliar parameters at the different sampling points by separating all of the leaves of each lettuce unit. For each leaf, we measured the length and width in the central portion of the leaf using an electronic digital caliper (#147; General Tools & Instruments LLC, Columbus, OH, USA), the leaf area using software (Easy Leaf Area software; University of California, Davis, CA, USA), as reported by Easlon and Bloom (2014), and the DM by weighing the leaves in an analytical balance after drying them for 72 h in paper bags in an oven at 72 °C. At 28 and 35 DAT, when the head lettuce was forming, the inner leaves that formed the head (hereafter referred to as head leaves) were counted and weighed separately from the outer leaves (external unfolded leaves).
Additionally, we evaluated whether the application of the product had any effect on the proportion of leaves that were sinks, sources, or transitioning between one stage and another at each sampling point. For this purpose, we referred to previous research of the transition from the sink to source stages for leaves (Dethloff et al. 2017; Fellows and Geiger 1974; Turgeon 1989). This transition usually starts soon after the leaf begins to unfold (Turgeon 1989) and occurs when the leaves reach 30% to 60% of their total expansion (Dethloff et al. 2017; Fellows and Geiger 1974). Based on this information, we relied on the leaf area measurements to track overall expansion and investigate the sink-to-source shift in lettuce leaves. At each sampling point, we used the highest leaf area value from the control treatment and considered it as 100%. Using this value as a 100% reference, we calculated the percentage of the leaf area for each leaf in both treatments. Leaves with a leaf area exceeding 60% were classified as source leaves, those with a leaf area below 30% were classified as sink leaves, and those with a leaf area between 30% and 60% were classified as transition leaves. Additionally, we also considered T2 leaves that exceeded the maximum area of the control (Fig. 3).
Based on the aforementioned parameters, including leaf area and biomass, we calculated growth indices that enable the functional analysis of the plant’s growth by observing the changes in biomass relative to leaf area over time. These indices were used to verify changes in the whole-plant biomass growth rate caused by the imposed treatments. The relative growth rate (RGR; increase in dry mass per unit plant dry mass and time; g·d−1), leaf area ratio (LAR; leaf area per unit total plant biomass; m2·g−1), net assimilation rate (NAR; DM gain per unit leaf area per unit time; g·m−2·d−1), specific leaf area (SLA; leaf area per unit leaf dry mass; m2·g−1), leaf mass ratio (LMR; ratio of leaf mass to plant mass; g leaf/g plant), root mass ratio (RMT; ratio of root mass to plant mass; g root/g plant), and root-to-shoot ratio were calculated.
Proteins, amino acids, reducing sugars (RS), total soluble sugars (TSS), starch, and sucrose were measured to assess whether the cytokinin B-Mo-based product had an effect on the flow of sugars from source to sinks through changes in the proportion of the total biomass stored in nonstructural C. Macromolecules were extracted from the samples (roots, outer leaves, and head leaves) by homogenizing 0.2 g of dry, ground, and sieved material in 10 mL of 0.1 M potassium phosphate buffer (pH = 7) and then subjected to a water bath at 40 °C for 30 min. The homogenate was centrifuged at 5000 gn for 10 min and the supernatant was collected to determine the levels of proteins, amino acids, RS, and TSS. For sucrose extraction, the supernatant was incubated with 30% KOH (Van Handel 1968). For starch extraction, the pellet was stored for resuspension with 200 mM potassium acetate buffer (pH = 4.8) (Zanandrea et al. 2009). The methods used for quantification were as follows: the anthrone reagent method (Dische 1962) for TSS, starch, and sucrose; the dinitrosalicylic acid method (Miller 1959) for RS; the ninhydrin method (Yemm et al. 1955) for amino acids; and the Bradford protein assay method (Bradford 1976) for proteins.
Statistical analyses were conducted using RStudio version 2023.06.0 (Posit, Boston, MA, USA). Normality was checked using the Shapiro-Wilk test. Because not all the variables were normally distributed, the homogeneity of variances between runs was checked using Bartlett’s and Fligner-Killeen’s tests for variables that were normally and non-normally distributed, respectively. To establish the significance of the effects of all factors (α = 0.05), data were analyzed using the Kruskal-Wallis one-way analysis of variance for growth and DM data and the Scheirer–Ray–Hare test for the nonstructural carbohydrates content, which has two sources of variation, the treatments (T1 and T2) and organs (roots, outer leaves, and head leaves). Because different individuals were destructively sampled at each sampling point, statistical differences between treatments were evaluated for each sampling point separately. The Dunn’s multiple comparison post hoc test with Bonferroni correction was used following a significant test. Because there was homogeneity between runs for most measurements (P < 0.05), both experimental runs were analyzed together.
According to the BBCH scale to monitor phenological stages, the leaf development that characterizes PGS 1 from 14 to 30 DAS showed that the application of the cytokinin B-Mo-based product had a statistically significant effect (P < 0.05) on the number of true unfolded leaves for most of the evaluated dates (Fig. 1C). On average, the T2 plants had 1 ± 0.4 more leaves than the T1 treatment plants. Regarding the head size from 30 to 50 DAS that characterizes PGS 4, the application of the cytokinin B-Mo-based product had a statistically significant effect (P < 0.05) on the head area of the plants. The area obtained by the control treatment at 50 DAS (504.93 cm2) was considered 100% of the expected head size for the cultivar. Thus, according to the scale, the typical size, form, and firmness of the head (SGS 9) was considered to reach 90% of that value. The head area values obtained were modeled over time and two curves were obtained, one for each treatment (Fig. 1D). The results indicated that the T2 plants had 43.12 cm2 more area, which corresponded to a 9% increase compared with that of the T1 plants at 50 DAS. Furthermore, the T2 plants reached SGS 9 at 42 DAS, whereas the T1 plants reached the same stage at 46 DAS. This suggests that reducing the growing cycle by 4 d would achieve a lettuce of the same size and form.
The application of the cytokinin B-Mo-based product had a statistically significant effect (P < 0.05) on the soil plant analysis development (SPAD) index at 14, 21, 28, and 35 DAT, with leaves from T2 showing higher SPAD index values than those of the leaves from the control treatment (Fig. 2A). Regarding the shoot and root length, the application of the cytokinin B-Mo-based product did not have a statistically significant effect (P < 0.05) on the shoot length at any of the sampling points (Fig. 2B). The application of the product only had a significant effect on root length at 21 DAT. The T2 plants had longer roots (+10.38 cm) than those of the control group (Fig. 2C). The application of the cytokinin B-Mo-based product had a statistically significant effect (P < 0.05) on the shoot, root, and total DM. Furthermore, T2 plants accumulated more DM in the shoot at 21 (+0.27 g), 28 (+0.3 g), and 35 (+0.9 g) DAT compared with that of the control group (Fig. 2D). Additionally, T2 plants accumulated more DM in the root at 14 (+0.13 g) and 28 (+0.1 g) DAT compared with that of the control group samplings in which root length was unaffected (Fig. 2D). The total DM had the same trend as that of the shoot DM, with significant statistical differences and T2 plants accumulating more total DM at 21 (+0.24 g), 28 (+0.39 g), and 35 (+0.83 g) DAT compared with that of the control group (Fig. 2F).
Citation: HortScience 60, 2; 10.21273/HORTSCI18281-24
Figure 3 illustrates the number of leaves and their respective leaf area for each treatment at each sampling point, with the 100% leaf area expressed relative to the maximum value observed in the control treatment. The 30% and 60% total leaf expansion thresholds were used as benchmarks to categorize leaves as source, sink, or transition leaves. Figure 4A presents the percentages resulting from this categorization. At 7 DAT, plants had an average of five leaves, with T1’s largest leaf measuring 6.4 cm2. Leaf categorization across both treatments stood at 20% sink leaves, 20% in transition, and 60% source, with T2 exhibiting one leaf over the 100% leaf area (Fig. 4A). By 14 DAT, the average number of leaves increased to 10 for both treatments and T1’s largest leaf area expanded to 24.36 cm2. Moreover, T1 had 10% sink leaves, 40% in transition, and 50% source, and T2 had 20% sink leaves, 30% in transition, and 50% source, with one leaf exceeding 100% area. At 21 DAT, the most pronounced differences emerged. On average, plants displayed 17 leaves, with T1’s largest leaf measuring 61.99 cm2. Leaf categorization for T1 was 30% sink leaves, 25% in transition, and 45% source, whereas T2 exhibited 10% sink leaves, 30% in transition, and 60% source (Fig. 4A), with five leaves exceeding 100% area. By 28 DAT, plants maintained 17 leaves on average, with T1’s largest leaf retaining a size of 132 cm2. Leaf categorization mirrored earlier stages, with 20% sink, 20% transition, and 60% source across both treatments. Finally, by 35 DAT, plants had an average of 19 leaves, with T1’s largest leaf retaining a size of 163.7 cm2. Sink leaves were present in equal proportion in both treatments (11%); T1 had 16% in transition and 74% source leaves, whereas T2 had 11% in transition and 79% source leaves, with eight leaves exceeding 100% area (Fig. 4A).
Citation: HortScience 60, 2; 10.21273/HORTSCI18281-24
Citation: HortScience 60, 2; 10.21273/HORTSCI18281-24
However, the application of the cytokinin B-Mo-based product had a statistically significant effect (P < 0.05) on the length and width of the leaves only at 21 DAT. For this sampling, T2 plants had longer (+0.7 cm) and wider (+0.9 cm) leaves compared with those of the control group (Fig. 4B, 4C). For the leaf area, the application of the product had a statistically significant effect at 21 (+7.5 cm2) and 35 (+8 cm2) DAT, with T2 plants having more surface area compared with that of the control group (Fig. 4D).
The application of the cytokinin B-Mo-based product did not have a statistically significant effect (P < 0.05) on the relative growth rate (Fig. 5A), which suggested that, for both treatments, the speed at which the plans were growing relative to their sizes was the same. With the same trend, the product also did not have a statistically significant effect on the leaf mass ratio (Fig. 5D), indicating that the proportion of the plant total biomass that was allocated to the leaves was similar between the treatments. However, the product application had a statistically significant effect (P < 0.05) on the net assimilation rate between 7 and 14 DAT (Fig. 5B). Compared with the NAR of the control treatment during this period, T2 had a higher NAR, indicating greater efficiency of those plants when converting available resources into biomass in the first week of growth. This may be related to the greater accumulation of biomass in the roots of T2 plants at 14 DAT (Fig. 2E). At 21 DAT, the application of the cytokinin B-Mo-based product had a statistically significant effect on the leaf area ratio and specific leaf area. Both parameters indicated a higher investment of T2 plants in leaf surface area in comparison with that of the control. This is supported by the higher values of leaf length, width, and area of the T2 plants at the same DAT (Fig. 4B–4D). The root-to-shoot ratio (Fig. 5F) was only affected by the application of the cytokinin B-Mo-based product at 35 DAT. The T1 plants had a higher root-to-shoot ratio compared with that of the T2 plants, indicating that T2 plants allocated more biomass to the shoot than to the roots. This is supported by the higher values of shoot DM of T2 plants at 35 DAT (Fig. 2D).
Citation: HortScience 60, 2; 10.21273/HORTSCI18281-24
Between 7 and 21 DAT, the protein and amino acid contents were not statistically influenced (P < 0.05) by any of the sources of variation (treatments or organs) or their interaction (Table 1). At 28 and 35 DAT, there were only statistically significant differences between the organs, without differences between the treatments or the interaction of both factors. At 28 DAT, the average protein content was higher in the head leaves (20.98 mg protein/g DM), followed by that of the outer leaves (11.04 mg protein/g DM) and roots (3.21 mg protein/g DM). At 35 DAT, the head leaves contained the highest average protein content (18.20 mg protein/g DM), followed by that of the outer leaves/T1 roots (7.52 mg protein/g DM) and T2 roots (3.17 mg protein/g DM) (Fig. 6A). Regarding the amino acid contents, similarly, at 28 DAT, the T1 head leaves had the highest average content (514.18 µmol aa/g DM), followed by that of the T2 head leaves/outer leaves (average, 347.21 µmol aa/g DM) and roots (average, 134.87 µmol aa/g DM). The trend was similar at 35 DAT, where the T1 head leaves had the highest average content (424.84 µmol aa/g DM), followed by that of the T2 head leaves/outer leaves (average, 363.61 µmol aa/g DM) and roots (average, 118.13 µmol aa/g DM) (Table 1).
Citation: HortScience 60, 2; 10.21273/HORTSCI18281-24
Regarding the RS contents, the statistical analysis revealed no significant differences between the treatments for all sample points. However, differences were observed between the organs. In general, between 7 and 21 DAT, the RS content was higher in the leaves than in the roots (Fig. 6). At 28 DAT, the T1 head leaves had the highest average content (214.51 µmol RS/g DM), followed by that of the T2 head leaves/T1 outer leaves (122.08 µmol RS/g DM), T2 outer leaves (71.90 µmol RS/g DM), and the roots (18.75 µmol RS/g DM). At 35 DAT, the average RS content was higher in the head leaves (293 µmol RS/g DM), followed by that in the outer leaves (100.96 µmol RS/g DM) and roots (21.59 µmol RS/g DM). Based on the analysis of TSS contents, there were significant statistical differences between the organs only at 7 and 14 DAT. For these sampling points, the TSS content was generally higher in the leaves than in the roots. At 21, 28, and 35 DAT, the treatments, organs, and interaction between the two sources of variation showed significant statistical differences. Specifically, at 21 and 28 DAT, the leaves of T2 plants had a higher TSS content (average, 192.99 µmol TSS/g DM) than that in the leaves of T1 plants (average, 116.80 µmol TSS/g DM). Similarly, at 35 DAT, the T2 plants’ head leaves had a higher TSS content (701.85 µmol TSS/g DM) than that of the T1 plants’ head leaves (479.95 µmol TSS/g DM). In general, the head leaves of the plants contained a higher TSS content than that of the outer leaves and roots (Fig. 6).
Similarly, the sucrose contents were statistically significantly different only between the organs at 7 and 14 DAT, with higher contents in the leaves compared with that in the roots. At 21 and 28 DAT, the treatments and organs showed statistically significant differences. At 35 DAT, all sources of variation and their interaction showed significant statistical differences. At 21 and 28 DAT, the T2 plants had a higher sucrose content in their leaves (average, 155.63 µmol Suc/g DM) compared with that in the T1 leaves (average, 100.72 µmol Suc/g DM). Additionally, at 28 DAT, the T2 plants had a higher sucrose content in their roots (50.39 µmol Suc/g DM) compared with that in the roots of T1 plants (30.75 µmol Suc/g DM). Furthermore, at 35 DAT, the T2 plants’ head leaves had a significantly higher sucrose content (415.05 µmol Suc/g DM) compared with that in the head leaves of T1 plants (306.59 µmol Suc/g DM). Regarding the starch contents, statistical differences were observed between the organs for all sampling points, with differences between the treatments only at 21 DAT. At this sampling point, the T1 plants had a higher starch content in their leaves (97.40 µmol·g−1 DM) compared with that in the leaves of the T2 plants (82.50 µmol·g−1 DM). For this variable, the starch content was higher in the head leaves, with similar values in the outer leaves and roots (Fig. 6).
Overall, the concentration of macromolecules in head leaves was higher than that in outer leaves and roots (Fig. 6). This trend was consistent across all the macromolecules studied. Furthermore, statistically significant differences were mainly observed between treatments 21 and 35 DAT. During this period, some of the foliar, growth, and DM accumulation variables also showed differences.
In this study, we assessed whether a commercial cytokinin-B-Mo-based product altered the source-to-sink relationship during the leaf development of lettuce plants and consequently impacted plant growth and quality. To achieve this, we addressed the tradeoffs in C allocation among different plant organs and processes by considering the spatiotemporal variations in growth rates, biomass allocation, and nonstructural carbohydrate contents. This provided an understanding of how the cytokinin B-Mo-based product affects the overall development of lettuce plants. Temporal changes addressed the dynamics observed at specific sampling points throughout the plant cycle from the seedling stage to harvest, whereas spatial variations included the dynamics among different plant organs.
The assessment of the sugar partitioning can be accomplished by analyzing the biomass distribution among plant organs and their actual distribution patterns (Poorter et al. 2012). The growth indices in this study illustrate biomass changes in relation to leaf area based on allocation fractions of leaves (outer and head leaves), roots, or the whole plant. These changes are likely associated with the complex network of source-to-sink dynamics regulated by the inter-relation and cross-talk between sugars and cytokinins (Wang et al. 2021). Cytokinins play a pivotal role in regulating parameters that affect source or sink strength, including C fixation, assimilation, partitioning of primary metabolites, and cell-cycle activity (Werner et al. 2008). Moreover, B, an essential micronutrient, has been identified as a direct regulator of meristem activity, cell proliferation, and differentiation, triggering the proper development of vascular plants (Pereira et al. 2021). Additionally, B has been recognized as a key player in the translocation of sugars from source to sinks (Dannel et al. 2002; Perica et al. 2001; Rehman et al. 2018; Stangoulis et al. 2010). In this study, growth indices and plant growth trends reflect how changes in allocation fractions are linked to the source-to-sink dynamics regulated by sugars and cytokinins, which are potentially influenced by the application of the cytokinin B-Mo-based product.
In our experiment, the rate of DM accumulation per unit of leaf area per unit of time (NAR) differed significantly by treatment during 7 to 14 DAT (Fig. 5). This suggests that T2 plants may have more efficiently converted available resources into biomass, possibly because of the higher root DM observed in this treatment at 14 DAT (Fig. 2E), as well as the greater number of true unfolded leaves in the days preceding head formation (Fig. 1C). The increased root biomass could have enhanced nutrient and water uptake, thus contributing to more rapid initial growth. However, despite a second application of the cytokinin B-Mo-based product midcycle, no significant differences between treatments were observed toward the end of the cycle. Additionally, the LMR did not vary between treatments at any sampling point, indicating that the allocation of biomass to leaves remained proportional to the total dry mass within each treatment throughout the cycle (Fig. 5D).
At 21 DAT, along with the second application of the cytokinin B-Mo-based product and the beginning of head formation, noticeable differences in leaf parameters were observed. The higher LAR (leaf area per unit total mass) and SLA (leaf area per unit leaf mass) in T2 plants suggest that these plants more effectively maximized leaf area relative to biomass, likely contributing to improved light capture and photosynthetic efficiency. This is further supported by the increased leaf size (area, width, and length) observed in T2 plants (Fig. 4). The LAR and LMR peaked between 21 and 28 DAT, indicating a period of rapid leaf expansion before stabilization As head formation progressed, leaf growth slowed, and DM accumulation became more prominent, leading to a gradual decline in LAR and SLA by 35 DAT as the plants shifted their resources toward biomass accumulation rather than further leaf expansion. Interestingly, despite these morphological differences, the RGR, calculated from the NAR, SLA, and LMR (Hilty et al. 2021; Osone et al. 2008; Shipley 2006), showed no statistical differences between treatments at any of the sampling points (Fig. 5). Both treatments experienced significant growth between 7 and 21 DAT that was driven by increases in size and leaf area. However, as leaf expansion ceased and biomass accumulation became the dominant growth process, the RGR stabilized, reflecting a shift in growth dynamics as the plants matured. This suggests that while the cytokinin B-Mo-based product may have influenced leaf development and morphology, resulting in larger leaves and overall plant size from 21 DAT onward, the proportional allocation of biomass within each treatment was conserved.
Significant variations in growth rates and DM accumulation observed during the second application of the cytokinin B-Mo-based product at 21 DAT may be linked to the development of a strong sink associated with head formation. Cytokinins have been linked to the establishment of local metabolic sinks and can influence physiological processes that determine sink strength (Werner et al. 2008). For example, cytokinins play a crucial role in various phases of the cell division cycle and act as key regulators of the balance between cell division and differentiation, which, in turn, affects organ shape and size (Wang et al. 2021). During the growth of sink tissues, sugar transport is facilitated by enzymes such as extracellular invertase and hexose transporters, which coordinate to ensure a steady carbohydrate supply. Cytokinins coregulate these enzymes, with one primary mechanism being the enhancement of sink potential by increasing the tissue’s capacity to import carbohydrates. Additionally, cytokinins stimulate extracellular invertase, which further enlarges sink size by promoting cell division through sugar signaling. Once induced by sugars, extracellular invertase establishes a feedback loop that amplifies and maintains the cytokinin signal, ensuring sustained regulation of the source–sink dynamics at the whole plant level (Roitsch and Ehness 2000). In parallel, B plays a direct role in regulating cell division and differentiation in the meristematic regions, mediating the developmental phase transitions of plants (Shireen et al. 2018). This suggests that during leaf development in T2 plants, particularly at the initiation of head formation (21 DAT), the interplay between cytokinins and B may influence cell cycle activity, leading to the formation of strong metabolic sinks. These changes in cell division and expansion may result in increases in both cell number and size, thus contributing to the larger leaf dimensions observed in T2 plants, as reflected by the increased leaf length (Fig. 4B), width (Fig. 4C), and area (Fig. 4D). This, in turn, led to higher values for LAR and SLA. Additionally, the higher proportion of source leaves compared with that of sink leaves or leaves in transition (Fig. 4A) likely contributed to these differences. The increase in root length for this period (Fig. 2C) could also be linked to the regulation of cell cycle and meristematic activity in the roots by B (Pereira et al. 2021).
After the second application of the cytokinin B-Mo-based product at 21 DAT, it was evident that some growth trends in the leaves and roots showed significant differences among treatments. Although some of these differences did not persist until the end of the cycle, the T2 plants continued to exhibit higher leaf area (Fig. 4A) as well as shoot (Fig. 2D) and total (Fig. 2F) DM accumulation until the end of the cycle. These differences led to reaching SGS 9 of the BBCH scale, which represents the typical head size, shape, and firmness in less time (Fig. 1). However, these findings may indicate that frequent applications might be necessary to sustain consistent growth trends, particularly after a strong sink has been established.
During its life cycle, a plant must face changes in C allocation associated with changes in the plant growth rates and competing C sinks for sugar availability (Durand et al. 2018; Wingler 2018). The development of new source and sink organs during ontogeny shifts the overall control of growth because changes in the number, size, and activity of plant organs alter the internal capacity of a plant to acquire and consume resources (White et al. 2016). These resources, mainly sugars, are regulated both temporally and spatially, thus exerting a crucial influence on plant growth and development (Lastdrager et al. 2014). Therefore, the source-to-sink transport of sugars is one of the major determinants of plant growth and regulates the C allocation across plant organs (Lemoine et al. 2013) as well as the developmental transitions along the plant cycle (Wingler 2018).
In terms of spatial distribution and differences between autotrophic (source leaves) and heterotrophic (sink leaves and roots) organs, for both treatments and for all the NSCs, proteins, and amino acids evaluated, there was a general trend of higher contents in the leaves than in the roots between 7 and 21 DAT. From 28 and 35 DAT, when the head leaves were also considered separate from the outer leaves, a trend of higher contents in the head leaves compared with that in the other organs was generally observed (Fig. 6). Based on the overall size of the leaves in the T1 treatment, between 7 and 21 DAT, the proportion of outer leaves categorized as source was comparable to the combined number of leaves categorized as sink and those in transition (Fig. 4A). Following the initiation of head formation, the number of new leaves as well as growth in length and width of the outer leaves ceased. This trend resulted in an increased number of leaves categorized as sources, whereas the head leaves continued to grow actively until the end of the cycle. The active growth and development of those leaves involved an increase in cell volume and cell division, which largely depend on the availability of carbohydrates for energy and biomass (Lastdrager et al. 2014). Therefore, the head leaves acted as a primary sink and demanded high levels of carbohydrates and nutrients to meet the building blocks needed to maintain the high metabolic rates, ensuring a high rate of assimilate catabolism (Roitsch and Ehness 2000).
For example, N, one of the highly required nutrients, is mainly used for growth in the form of proteins, with a notably high requirement in leaves because of the assembly and maintenance of the complex enzyme-rich photosynthetic machinery (White et al. 2016). The N availability regulates the uptake and storage of C because a high N status increases the rate of C acquisition in photosynthesis and upregulates C sinks. Furthermore, N enhances the assimilation of nitrate by enzyme nitrate reductase (NR), thereby upregulating N source and sink activity (White et al. 2016). Additionally, NR plays a key role in inorganic N acquisition and assimilation (Coelho and Romão 2015; Hille et al. 2011). The Mo cofactors participate in the active site of NR, which suggests that Mo directly affects the NR molecule and, thus, the nitrate assimilatory pathway (Kovács et al. 2015; Liu et al. 2017; Rana et al. 2020). Although some studies have reported that the exogenous application of Mo results in higher contents of N and N-related products in sink tissues such as fruits and seedlings (Liu et al. 2017). In our case, the plants that received cytokinin B-Mo-based product application did not show significant differences in the contents of proteins and amino acids of the organs (Table 1).
Regarding the temporal scale, throughout the crop cycle, there was a trend of increasing protein and amino acid values for the outer leaves and head leaves, while the values in the roots remained relatively constant. Conversely, RS, TSS, sucrose, and starch demonstrated a trend of higher values at 7 and 14 DAT, followed by a noticeable decrease, especially in the outer leaves, from 21 DAT until the end of the cycle and an increase in the head leaves. This trend could be attributed to these leaves functioning as a strong sink, with a significant capacity for the import of photoassimilates, as discussed. Following the second application of the cytokinin B-Mo-based product at 21 DAT and the head formation, statistical differences in the contents of TSS, sucrose, and starch, particularly in the outer leaves and head leaves, were observed between treatments (Table 1).
In actively growing tissues, such as the head leaves, the distribution of photoassimilates is under strict developmental control (Werner et al. 2008). Sucrose transported into the sink tissue can be cleaved by sucrose synthases or invertases (Koch 2004). Invertases are particularly active in these actively growing tissues during sink initiation and expansion growth and play a crucial role in directing the allocation and intracellular distribution of various sugars and sugar-derived metabolites (Lastdrager et al. 2014; Roitsch and Ehness 2000; Tiessen and Padilla-Chacon 2013; Werner et al. 2008). Cytokinins have been found to upregulate the expression of genes encoding cell wall invertases (Balibrea Lara et al. 2004; Ehness and Roitsch 1997). Furthermore, scientific evidence has indicated that the induction of extracellular invertase is one of the molecular prerequisites for various cytokinin responses, including the provision of carbohydrates for growth and stimulation of cell division (Roitsch and Ehness 2000). The increase in sink size induced by cytokinins caused by cell division requires an investment of building blocks and energy, thus making carbohydrate availability an essential limiting factor. This highlights the significant link between cytokinins, sugar status, and the initiation of cell division (Guo et al. 2023; Roitsch and González 2004; Tiessen and Padilla-Chacon 2013).
Furthermore, exogenous applications of B have also been linked to increased invertase and sucrose synthase activity (Sahu et al. 2022). In addition to the increased activity of these enzymes, B plays an important role in metabolizing carbohydrates and enhancing transportation of assimilates into sink tissues through the formation of soluble B-complexes, increasing phloem sugar concentrations that fuel proper growth and development of the sink tissues (Hu et al. 1997; Pommerrenig et al. 2019; Stangoulis et al. 2010). These B-complexes facilitate the “shuttle” of compounds across the plasma membrane, resulting in the efflux of sugars in the cytoplasm (Dannel et al. 2002; Perica et al. 2001).
The increased levels of TSS and sucrose in the outer leaves at 21 and 28 DAT as well as in the head leaves at 35 DAT of T2 plants (Table 1) could be attributed to the impact of the exogenous application and interplay between cytokinins, B, and Mo. This increased sugar content suggests that sucrose was either synthesized more or loaded with higher efficiency into the phloem in T2 plants. For instance, this interplay could have promoted increased invertase activity, leading to increased sucrose cleavage at the site of phloem unloading. The cleavage products were then metabolized in the sink tissues and organs, ultimately resulting in increased sucrose and TSS contents in the T2 plants. Therefore, the application of the cytokinin B-Mo-based product may have enhanced the supply and distribution of carbohydrates, stimulated cell division, and ultimately contributed to the improved growth and accumulation of DM (Fig. 2), reaching a marketable and high-quality plant within a shorter timeframe (Fig. 1).
Our research of the use of a commercial product to strengthen the source-to-sink relationship during leaf development of lettuce plants offers valuable insights into nonstructural carbohydrate metabolism and the roles of cytokinin, B, and Mo in plant growth. Treated plants not only reached marketable size faster but also exhibited greater leaf surface area investment and improved the efficiency of converting assimilates into biomass. Cytokinin likely played a key role in promoting cell division and expanding sink size, increasing the tissue’s capacity to import carbohydrates by stimulating enzymes like extracellular invertase, which drives sugar metabolism in growing tissues. This process likely established a feedback loop that amplified the cytokinin signal, allowing for sustained regulation of source–sink dynamics. Additionally, B appeared to support cell division and differentiation in meristematic regions, contributing to developmental transitions during key growth stages such as head formation. The interaction between these active ingredients likely enhanced sink strength while also improving carbohydrate allocation to growing tissues, resulting in larger leaf dimensions, increased LARs, and SLAs in treated plants at some sampling points. These findings highlight the complex ways in which these components work together to promote efficient growth and resource utilization, ultimately leading to the production of high-quality lettuce plants within a shorter timeframe. Factors such as enzymatic activities and gene expression, including the role of marker enzymes like cell wall invertases, remain valuable targets for future research to validate and deepen our physiological understanding of these processes. Moreover, incorporating sensory analyses in future studies could provide insights into whether the increased sugar content impacts the taste profile of the lettuce, contributing to both product quality and consumer preference.
Monitoring of the phenological stage of Lactuca sativa cv. Rex plants using the Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie (BBCH) scale. (A) Description of the principal growth stage (PGS) and secondary growth stage (SGS) as defined by the BBCH scale. (B) Overhead image of plants used to determine head area with ImageJ software. (C) Progression of newly unfolded leaves from 14 to 30 d after sowing (DAS) in response to the application of Sugar Mover® Premier or the untreated control. (D) Proportional head area over time in response to the application of Sugar Mover® Premier or the untreated control expressed as a percentage of the final area (504 cm2) at harvest for the untreated control. The horizontal dotted line (- - -) marks the point when plants reach 90% of their final area, representing typical size, shape, firmness, and SGS 9 on the BBCH scale. Vertical dotted lines indicate the time (DAS) when each treatment reached SGS 9. Asterisks denote statistical differences according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis. ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
(A) Soil plant analysis development (SPAD) index, (B) shoot length (SL), (C) root length (RL), (D) shoot dry matter (SDM), (E) root dry matter (RDM), and (F) total dry matter (TDM) of Lactuca sativa cv. Rex plants in response to the application of Sugar Mover® Premier or untreated control. Statistical differences were determined according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis, with P ≤ 0.05 indicating significance (n = 24). ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. DAT = days after transplanting to deep-water culture systems.
Number of leaves and corresponding leaf area (cm2) of Lactuca sativa cv. Rex plants in response to the application of Sugar Mover® Premier or the untreated control at 7 DAT (A), 14 DAT (B), 21 DAT (C), 28 DAT (D), and 35 DAT (E). The letter “L” denotes the leaf number. The darker dashed line at the top of each graph indicates the maximum leaf area achieved by the control treatment at each sampling point set at 100% leaf expansion. The two lighter dashed lines represent 60% and 30% leaf expansion, respectively. Leaves with more than 60% leaf expansion were classified as source leaves, whereas those with less than 30% were categorized as sink leaves. Leaves exhibiting 30% to 60% leaf expansion were designated as transition leaves. Categorization based on leaf expansion was performed as described by Turgeon (1989) and Fellows and Geiger (1974). DAT = days after transplanting to deep-water culture systems.
Foliar parameters of Lactuca sativa cv. Rex leaves in response to the application of Sugar Mover® Premier or the untreated control. (A) Frequency of leaves categorized as sinks, sources, or in transition between these stages in untreated control plants (left bar) and treated plants (right bar). (B) Leaf length (LL; cm), (C) leaf width (LW; cm), and (D) leaf area (LA; cm2). Statistical differences were determined according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis, with P ≤ 0.05 indicating significance (n = 24). ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. DAT = days after transplanting to deep-water culture systems.
Growth indices calculated based on the growth and dry matter parameters of Lactuca sativa cv. Rex plants. (A) Relative growth rate (RGR; increase in dry mass per unit plant dry mass and time; g·d−1). (B) Net assimilation rate (NAR; dry matter gain per unit leaf area per unit time; g·m−2·d−1). (C) Leaf area ratio (LAR; leaf area per unit total plant biomass; m2·g−1). (D) Leaf mass ratio (LMR; the ratio of leaf mass to plant mass; g leaf/g plant). (E) Specific leaf area (SLA; leaf area per unit leaf dry mass; m2·g−1). (F) Root-to-shoot ratio (RSR). Each boxplot represents the average of 12 values (n = 12). Statistical differences were determined according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis, with P ≤ 0.05 indicating significance. ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. DAT = days after transplanting to deep-water culture systems. Sampling points = 7, 14, 21, 28, and 35 d after treatment impositions.
Proportional variation in the contents of protein, amino acids, reducing sugars (RS), total soluble sugars (TSS), sucrose, and starch in the roots, outer leaves, and head leaves of Lactuca sativa cv. Rex across treatments at each sampling point. Within each sampling point, the left bar represents the control treatment, and the right bar represents the Sugar Mover® Premier treatment. DAT, days after transplanting to deep-water culture systems. T1 = control; T2 = application of the cytokinin B-Mo-based product. For detailed values and statistical analysis of each macromolecule, refer to Table 1.
Contributor Notes
This work was supported by the Floriculture and Nursery Research Initiative (project award no. 5082-21000-018-00D) from the US Department of Agriculture Agricultural Research Service and the Multistate Project (project award CONS 01022, accession number 102637) from the US Department of Agriculture National Institute of Food and Agriculture.
R.E.R. is the corresponding author. E-mail: rosa.raudales@uconn.edu.
Monitoring of the phenological stage of Lactuca sativa cv. Rex plants using the Biologische Bundesanstalt, Bundessortenamt und Chemische Industrie (BBCH) scale. (A) Description of the principal growth stage (PGS) and secondary growth stage (SGS) as defined by the BBCH scale. (B) Overhead image of plants used to determine head area with ImageJ software. (C) Progression of newly unfolded leaves from 14 to 30 d after sowing (DAS) in response to the application of Sugar Mover® Premier or the untreated control. (D) Proportional head area over time in response to the application of Sugar Mover® Premier or the untreated control expressed as a percentage of the final area (504 cm2) at harvest for the untreated control. The horizontal dotted line (- - -) marks the point when plants reach 90% of their final area, representing typical size, shape, firmness, and SGS 9 on the BBCH scale. Vertical dotted lines indicate the time (DAS) when each treatment reached SGS 9. Asterisks denote statistical differences according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis. ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
(A) Soil plant analysis development (SPAD) index, (B) shoot length (SL), (C) root length (RL), (D) shoot dry matter (SDM), (E) root dry matter (RDM), and (F) total dry matter (TDM) of Lactuca sativa cv. Rex plants in response to the application of Sugar Mover® Premier or untreated control. Statistical differences were determined according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis, with P ≤ 0.05 indicating significance (n = 24). ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. DAT = days after transplanting to deep-water culture systems.
Number of leaves and corresponding leaf area (cm2) of Lactuca sativa cv. Rex plants in response to the application of Sugar Mover® Premier or the untreated control at 7 DAT (A), 14 DAT (B), 21 DAT (C), 28 DAT (D), and 35 DAT (E). The letter “L” denotes the leaf number. The darker dashed line at the top of each graph indicates the maximum leaf area achieved by the control treatment at each sampling point set at 100% leaf expansion. The two lighter dashed lines represent 60% and 30% leaf expansion, respectively. Leaves with more than 60% leaf expansion were classified as source leaves, whereas those with less than 30% were categorized as sink leaves. Leaves exhibiting 30% to 60% leaf expansion were designated as transition leaves. Categorization based on leaf expansion was performed as described by Turgeon (1989) and Fellows and Geiger (1974). DAT = days after transplanting to deep-water culture systems.
Foliar parameters of Lactuca sativa cv. Rex leaves in response to the application of Sugar Mover® Premier or the untreated control. (A) Frequency of leaves categorized as sinks, sources, or in transition between these stages in untreated control plants (left bar) and treated plants (right bar). (B) Leaf length (LL; cm), (C) leaf width (LW; cm), and (D) leaf area (LA; cm2). Statistical differences were determined according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis, with P ≤ 0.05 indicating significance (n = 24). ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. DAT = days after transplanting to deep-water culture systems.
Growth indices calculated based on the growth and dry matter parameters of Lactuca sativa cv. Rex plants. (A) Relative growth rate (RGR; increase in dry mass per unit plant dry mass and time; g·d−1). (B) Net assimilation rate (NAR; dry matter gain per unit leaf area per unit time; g·m−2·d−1). (C) Leaf area ratio (LAR; leaf area per unit total plant biomass; m2·g−1). (D) Leaf mass ratio (LMR; the ratio of leaf mass to plant mass; g leaf/g plant). (E) Specific leaf area (SLA; leaf area per unit leaf dry mass; m2·g−1). (F) Root-to-shoot ratio (RSR). Each boxplot represents the average of 12 values (n = 12). Statistical differences were determined according to the Kruskal-Wallis rank-sum test with Dunn’s post hoc analysis, with P ≤ 0.05 indicating significance. ns = nonsignificant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. DAT = days after transplanting to deep-water culture systems. Sampling points = 7, 14, 21, 28, and 35 d after treatment impositions.
Proportional variation in the contents of protein, amino acids, reducing sugars (RS), total soluble sugars (TSS), sucrose, and starch in the roots, outer leaves, and head leaves of Lactuca sativa cv. Rex across treatments at each sampling point. Within each sampling point, the left bar represents the control treatment, and the right bar represents the Sugar Mover® Premier treatment. DAT, days after transplanting to deep-water culture systems. T1 = control; T2 = application of the cytokinin B-Mo-based product. For detailed values and statistical analysis of each macromolecule, refer to Table 1.
