https://orcid.org/0009-0004-4745-0663
Figures
Abstract
Cauliflower is a crop with intricate developmental transitions influenced by both external and internal cues. Plant growth regulators (PGRs) play a key role in developmental transitions and plant responses to environmental factors. The present study aimed to investigate the endogenous levels of gibberellins (GAs) (i) at six different developmental stages and (ii) at four time points in four varieties of cauliflower using high-performance liquid chromatography (HPLC). The Pusa Ashwini, Pusa Sharad, Pusa Shukti, and Pusa Snowball Kt-25 varieties represent all four thermosensory-based maturity groups: early (20–27 °C), mid-early (15–20 °C), mid-late (12–16 °C), and late or snowball (10–16 °C), respectively. GA3 content was highest in Pusa Shukti (4.020 ppm) and lowest in Pusa Ashwini (3.091 ppm). A higher endogenous GA3 concentration was recorded at the bolting stage (4.118 ppm), seedling stage (4.057 ppm), and curd initiation stage (3.722 ppm), suggesting its role in stalk elongation. The difference in GA3 content was significant between curd (3.613 ppm) and leaf tissues (2.947 ppm) at the full curd stage and nonsignificant between stalk (3.948 ppm) and leaf tissues (4.118 ppm) at the bolting stage. Regarding the time points, the GA3 content was highest in Pusa Sharad (4.311 ppm) and lowest in Pusa Ashwini (2.990 ppm). GA3 content showed a significant positive correlation with duration to crucial developmental transitions, namely the curd initiation stage, the full curd stage, and the bolting stage. The study highlights the role of endogenous gibberellins in plant development and suggests their potential for benefiting seed production.
Citation: Luhana S, Singh S, Nagar S, Chandel A, Varotariya K, Mangal M, et al. (2025) Understanding the changes in endogenous GA3 in relation to developmental transitions in cauliflower (Brassica oleracea var. botrytis L.). PLoS One 20(6): e0321599. https://doi.org/10.1371/journal.pone.0321599
Editor: Kuldeep Tripathi,, NBPGR: National Bureau of Plant Genetic Resources, INDIA
Received: January 16, 2024; Accepted: March 8, 2025; Published: June 24, 2025
Copyright: © 2025 Luhana et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: This work was supported by the Indian Council of Agricultural Research (ICAR), New Delhi, through the CRP Hybrid Technology project (Cauliflower: CRPHT-142-12-F). SL received a fellowship for the master's program from ICAR, New Delhi, through the Graduate School, ICAR-Indian Agricultural Research Institute, New Delhi. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
For successful reproduction and genetic continuation, it is essential for plants to transition from the vegetative to the reproductive phase and produce flowers and seeds. Floral transition depends on the plant’s response to internal (endogenous) and external (environmental) cues. In the model plant Arabidopsis, extensive studies have revealed six major pathways deciding flowering time: photoperiod, vernalization, gibberellins, thermosensory (ambient temperature), age, and autonomous [1]. Gibberellins (GAs) promote flowering by upregulating the expression of floral integrators, such as LEAFY (LFY) and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), via DELLA protein-regulated mechanisms [2,3]. GAs govern a number of processes throughout the plant life cycle, including seed germination, stem and leaf growth, trichome development, flowering time, and vegetative and reproductive development [4]. The role of GAs in flowering time regulation has been extensively studied for several years, and it has been proven that flowering defects occur when there is a loss of function in various components of GA biosynthesis and signalling transduction [5]. In contrast, early flowering is induced by exogenous GAs or by elevated endogenous GA levels and enhanced GA signalling [5]. However, the effect of GA being either promoting, inhibiting, or neutral is also dependent on the plant species [6]. Cauliflower (Brassica oleracea var. botrytis L.) is a key member of the Cole group of vegetable crops and is cultivated in almost 97 countries on an accumulated area of 1.378 million hectares [7]. Cauliflower originated in the Mediterranean region and spread to different parts of the world, including India. Adaptational changes led to the evolution of different ecotypes of cauliflower, such as Originals, Northern, Cornish, Roscoff, Angers, Snowball, and tropical or Indian cauliflower. These evolutionary changes mostly affected the genomic regions associated with developmental transitions [8], which could also be the plant growth regulator (PGR) pathways [6]. Duclos and Björkman (2015) identified prominent GAs in cauliflower as GA8, GA29, GA1, GA20, and GA19, and in broccoli as GA8, GA29, GA1, and GA20 [6]. It is considered that cauliflower directly evolved from broccoli through stepwise domestication involving major gene mutations [9]. The absence of GA19, an immediate precursor to GA20, in broccoli suggests a possible divergence in the GA biosynthesis pathway between these two B. oleracea varieties during the domestication process.
The edible portion of cauliflower, i.e., the curd, is composed of arrested prefloral fleshy inflorescence meristems, marking the vegetative-to-generative transition in two phases. First, the transition from vegetative to inflorescence meristem occurs, followed by elongation of curd, known as bolting, under congenial conditions, leading to the transition from inflorescence meristem to floral primordium. However, these developmental transitions rely on genotypic responses to environmental conditions. Indian cauliflower has four maturity groups that respond to different temperature regimens for developmental transitions [10].
In Brassica vegetables, numerous studies have indicated that the application of exogenous GA3 induces early bolting and flowering, as well as elongation of stems and stalks [6,11–16], suggesting its potential impact on the endogenous hormone system. It also enhances morphological traits related to vegetative growth, curd, and seed yield in cauliflower [17–19]. The pathway of GA biosynthesis during the production of bioactive end products divides into two branches at GA12: one involves hydroxylation at C-13, known as the 13-hydroxylation pathway, resulting in the formation of 13-hydroxylated GAs such as GA1, GA5, and GA3, while the second, i.e., the non-13-hydroxylation pathway, results in the formation of GA4 and GA7 [20]. Early studies reported endogenous GAs from both pathways in cabbage shoots [21]. In cauliflower, the application of GA4+7 (GAs from the non-13-hydroxylation pathway) was shown to promote curd initiation [22,23], while a study analysing endogenous GA1 (GA from the 13-hydroxylation pathway) identified it as a possible factor governing inflorescence stalk elongation [11]. However, a recent study in cauliflower reported that endogenous GAs from both the early 13-hydroxylation pathway (specifically, GA1, GA3, precursors, and inactive metabolites) and the non-13-hydroxylation pathway (GA4) are evident in the inflorescence meristems of cauliflower, and exogenous application of both GA3 and GA4+7 shortens the vegetative period and accelerates the vegetative to reproductive transition [6]. Therefore, in addition to its exogenous applications, GA3 also serves endogenously as a crucial factor in the developmental physiology of cauliflower. To date, only a few studies on cauliflower have examined endogenous hormones, primarily focusing on reproductive development [6,11]. A notable gap remains in understanding how the concentration of endogenous GA3 varies among the different thermo-sensitive maturity groups and influences different developmental transitions. Therefore, the present study was designed to investigate the natural fluctuations in endogenous GA3 concentrations across different developmental transitions and time frames under ambient temperature conditions without any external treatment. To explore the potential role of GA₃ in temperature resilience and its implications for advancing cauliflower improvement for broader production plasticity, we also investigated the association between endogenous GA₃ dynamics and phenological transitions. This study included four maturity groups of cauliflower, namely, early, mid-early, and mid-late groups indigenous to India, as well as the late or snowball group.
Materials and methods
Plant material and phenological observations
Four varieties, one from each maturity group: Pusa Ashwini (early), Pusa Sharad (mid-early), Pusa Shukti (mid-late), and Pusa Snowball Kt-25 (PSB Kt-25) (late), were used in the study. The present study included two sowing-time points: (i) for the first experiment (i.e., stage-specific changes in GA content in different thermosensory groups) on 30 August 2022; and (ii) for the second experiment (i.e., changes in GA content at different growth time points) on 30 October 2022. The sowing was done in 12-inch-high earthen pots filled with a mixture of farm soil and vermicompost (2:1 ratio). One-month-old seedlings were subsequently transplanted into raised beds prepared inside the net house (a 40-mesh nylon net). The crop was raised following recommended cultivation practices [24]. Varieties were observed for critical developmental transitions, namely, the young stage (8 leaves) (YS), the adult stage (12 leaves) (AS), the curd initiation (CI) stage, the full curd (FC) stage, and the bolting stage (BS). Stalk length (SL) was measured at the full flowering stage, with an exception for PSB Kt-25, which bolted but did not flower; in this case, the stalk length at the full bolting stage was considered. The daily temperature data of the experimental site during the trial crop period were obtained from the Agro-Met Advisory, Division of Agricultural Physics, ICAR-IARI, New Delhi.
Determination of GA3 content
To ascertain whether the changes in GA3 content are attributed to developmental transitions or temporal factors, the GA3 content was determined in two distinct experiments. First, the GA3 content was analysed at six crucial developmental transitions: seedling (4 leaves) stage (SS), young stage (YS), adult stage (AS), curd initiation (CI) stage, full curd (FC) stage, and bolting stage (BS) in two replications. For the full curd and bolting stages, in addition to leaf tissues, GA3 content was also determined in curd and stalk tissues, respectively. These six developmental transitions in the four varieties are depicted in Fig 1. Second, the GA3 content in leaf tissues was analysed at four time points: 35, 55, 75, and 95 days after transplanting (DAT), with two replications. Samples were collected from mature leaves in the evening and stored at -80 °C until further processing.
Preparation of samples for GA3 analysis by high-performance liquid chromatography (HPLC)
The GA3 samples were prepared following the protocol described by [25] and [26] with minor modifications (S1 Fig). Each 2-gram sample was crushed with liquid nitrogen and homogenized with 10 ml of cold sodium phosphate buffer (0.05 M, pH 7.5) containing 0.02% sodium diethyldithiocarbamate, an antioxidant. After overnight shaking at 4 °C and 150 rpm, the prepared extracts were centrifuged at 10,000 rpm for 10 minutes at 4 °C. The resulting supernatants were collected and adjusted to a final volume of 10 ml with sodium phosphate buffer. The samples were then partitioned using a separating funnel with 5 ml of diethyl ether, and the aqueous phase was collected. The pH of the resulting aqueous phase was adjusted to 2.5 using 1 N HCl. The aqueous phase was partitioned twice with 5 ml of petroleum ether, with each aqueous phase being collected and the ether phase discarded. The aqueous phase was further partitioned three times with 5 ml of diethyl ether. For the first two times, the aqueous phase was collected, and in the last step, the ether phase was taken and filtered through sodium sulphate crystals using Whatman Number 1 filter paper (Whatman plc, UK). The filtrate was collected and left overnight for lyophilization in a small beaker. The next day, the beaker was rinsed with 1 ml of 26% acetonitrile (mobile phase) prepared using HPLC water, filtered through a polyvinylidene difluoride (PVDF) membrane filter (diameter: 13 mm, filtration rate: 0.45 µm), and stored at -20 °C for the determination of GA3.
HPLC protocol
The GA3 analysis in the samples was conducted using HPLC (Agilent 1100/1200 series, Agilent Technologies, Santa Clara, CA, USA) at the Division of Plant Physiology, ICAR-IARI, New Delhi. The separation was achieved on a C18 column (Agilent Eclipse XDB-C18, 5 µm pore size, 4.6 × 200 mm; Santa Clara, CA, USA) maintained at 30 °C. The mobile phase consisted of acetonitrile: water (26:74; v/v, pH 3.5) filtered through a PVDF membrane filter. The flow rate was maintained at 0.8 mL/min, and a 20 µL sample of the extract was injected into the column. The wavelength for the determination was 206 nm for UV monitoring and 360 nm for recording. GA3 served as an external standard (0.1, 0.5, 1, 5 and 10 mg/L) for identifying the hormone in plant samples. The peak area was utilized to determine the hormone concentration using the formula: GA3 concentration (ppm) = peak area/graph factor. The graph factor was obtained from the standard graph (S2 Fig).
Statistical analysis
Statistical analysis for GA3 content data was performed using a two-factor randomized block design (RBD) to determine the interactive effect (I) of genotype (G) and developmental transitions (DT) or plant parts (PP) or time points (TP) on the endogenous GA3 concentration and a one-factor RBD for phenological observations by using RStudio packages agricolae and doebioresearch. The normality of the distribution of the data was confirmed with the Shapiro-Wilk normality test. Duncan’s multiple range test (DMRT) at the 5% significance level was used to identify significant differences among treatment means. Replication-wise data is provided in the Supporting Information (S1–S5 Tables). Changes over the seedling stage and 35 DAT were computed using MS Excel. Graphical representation and correlation analysis were accomplished using OriginPro 2023b.
Results
Phenological observations
Concerning the 30 August 2022 sowing, Pusa Ashwini (early) and Pusa Sharad (mid-early) were the earliest for the CI stage at 104 days (Table 1). Pusa Ashwini progressed quickly, with the FC stage at 115 days and bolting at 124 days, while Pusa Sharad had the CI stage at 141 days and the BS at 152 days. Pusa Shukti (mid-late) and PSB Kt-25 (late) attained the CI stage at 117 and 142 days, respectively. SL was highest in Pusa Shukti (78.67 cm) and lowest in PSB Kt-25 (15.33 cm). For 30 October 2022 sowing, 35 DAT, all genotypes were at the YS (8-leaf stage). Pusa Ashwini reached the FC stage without attaining the proper juvenile or adult stage (AS) by 55 DAT (Table 2). By 95 DAT, Pusa Ashwini exhibited flower termination, Pusa Sharad exhibited bolting initiation, and Pusa Shukti and PSB Kt-25 exhibited CI stage. The temperature influence on the developmental transitions of both sowings is depicted in Fig 2.
(A) Developmental transitions for the 30 August 2022 sowing. (B) Developmental transitions and plant developmental stage corresponding to time points for the 30 October 2022 sowing. (Source of temperature data: Division of Agricultural Physics, ICAR-IARI) SS - Seedling Stage, YS - Young Stage, AS - Adult Stage, CI - Curd Initiation, FC - Full Curd, BS - Bolting Stage.
Endogenous GA3 content at developmental transitions
Both genotype and developmental transitions, as well as their interactive effect, showed statistically highly significant differences at the 0.1% level (Table 3).
The endogenous GA3 content in the leaves was highest at the bolting stage (4.118 ppm), which was on par with the seedling stage (4.057 ppm) but significantly higher than the GA3 content at the full curd stage (2.988 ppm), young stage (3.339 ppm), adult stage (3.507 ppm), and curd initiation stage (3.722 ppm) (Table 4). Among the varieties, the maximum GA3 content was observed in Pusa Shukti (4.020 ppm), and the minimum was observed in Pusa Ashwini (3.091 ppm). The GA3 content in the mid-late group variety Pusa Shukti was comparable to the late group variety PSB Kt-25 (3.994 ppm). The mid-early genotype Pusa Sharad (3.383 ppm) exhibited a higher GA3 content than the early group genotype Pusa Ashwini (3.091 ppm). The interactive effect revealed maximum GA3 content in PSB Kt-25 at the seedling stage (5.088 ppm), followed by Pusa Shukti at the bolting stage (4.462 ppm) and its adult stage (4.443 ppm).
Except for Pusa Sharad, a reduction in the GA3 content from the seedling to the young stage was observed for the remaining three varieties (Fig 3A). At the adult stage, the GA3 content increased in Pusa Ashwini (2.791 ppm) and Pusa Shukti (4.443 ppm), decreased in PSB Kt-25 (3.235 ppm), and remained almost the same in Pusa Sharad (3.558 ppm). During curd initiation, GA3 content increased significantly in Pusa Ashwini (3.339 ppm) and in the PSB Kt-25 (3.771 ppm), remained nearly the same in Pusa Sharad (3.596 ppm), and slightly decreased in Pusa Shukti (4.184 ppm). At the full curd stage, the GA3 content decreased in all the varieties, with a significant reduction in Pusa Ashwini (1.794 ppm) and Pusa Sharad (2.838 ppm). The bolting stage showed a significant increase in GA3 content in all the tested varieties, with the maximum content observed in Pusa Shukti (4.462 ppm), followed by PSB Kt-25 (4.292 ppm), Pusa Ashwini (4.227 ppm), and Pusa Sharad (3.490 ppm).
(A) GA3 content (ppm) at developmental transitions. (B) GA3 content (ppm) at time points. DAT - Days After Transplanting.
At the full curd stage, notable differences in GA3 content were observed in both the leaf (2.947 ppm) and curd (3.613 ppm) sections, a pattern consistent across varieties except for PSB Kt-25 (late) (Table 5). Among these varieties, Pusa Sharad had the highest GA3 content (3.729 ppm), followed closely by Pusa Shukti (3.343 ppm), while Pusa Ashwini had the lowest concentration (2.731 ppm). Specifically, within curd tissues, Pusa Sharad (mid-early) had the maximum content (4.620 ppm), while Pusa Shukti had the minimum content (2.765 ppm). Interestingly, at the bolting stage, no significant difference was observed in the leaf (4.118 ppm) and stalk (3.948 ppm) portions (Table 5). Within the stalk tissues, Pusa Shukti had the highest GA3 content (4.450 ppm), whereas Pusa Sharad displayed the lowest concentration (3.246 ppm). Significant differences were observed in the curd (3.572 ppm) and stalk (3.948 ppm) portions collected from the full curd and bolting stages, respectively. Except for Pusa Ashwini, the tested varieties showed significant differences in GA3 content in the curd and stalk portions sampled from their respective stages.
Two-factor RBD analysis of variance (ANOVA) for genotypes and plant parts is provided in Table 3.
The HPLC chromatograms for one replication are presented in S3 Fig.
Changes in GA3 content over the seedling stage
In the case of Pusa Ashwini, except during the bolting stage, the GA3 content consistently remained lower than that in the seedling stage at all the other stages (Fig 4A). The most significant reduction, amounting to -56.41%, was noted at the full curd stage, which then showed a slight increase of 2.72% over the seedling stage upon entering the bolting stage. In contrast, except for a decrease of -14.45% at the full curd stage, the GA3 content in Pusa Sharad was higher at all the other stages. The highest increase over the seedling stage was observed at the curd initiation stage (8.42%), followed by the adult stage (7.28%). Pusa Shukti maintained a higher GA3 content than that in the seedling stage, except during the young stage, where there was a decrease of -8.24%. The most notable increase was observed during the bolting stage (20.37%), followed by the adult stage (19.86%). In PSB Kt-25, the GA3 content remained consistently lower than that in the seedling stage at all developmental stages. The most significant reduction was observed in the adult stage (-36.43%), followed by the full curd stage (-33.19%). The bolting stage (-15.66%) and the young stage (-17.92%) showed comparatively lower reductions.
(A) Change (%) over seedling stage. (B) Change (%) over 35 days after transplanting (DAT).
Endogenous GA3 content at four time-points
ANOVA revealed statistically highly significant differences for genotype (p<0.001), time points (p<0.01), and their interactive effect (p<0.001) (Table 3).
At 35 DAT, Pusa Ashwini (3.352 ppm) and Pusa Sharad (3.372 ppm) had similar GA3 concentrations, while Pusa Shukti (4.057 ppm) and PSB Kt-25 (4.508 ppm) had higher levels (Table 6). At 55 DAT, there was a significant decrease in the GA3 content in Pusa Ashwini (2.453 ppm) and Pusa Shukti (3.374 ppm), while Pusa Sharad showed a significant increase (4.784 ppm) (Fig 3B). PSB Kt-25 exhibited a slight decrease in GA3 content (4.276 ppm). At 75 DAT, the GA3 content significantly decreased in Pusa Sharad (4.359 ppm) and PSB Kt-25 (3.168 ppm), while it remained relatively constant in Pusa Ashwini (2.748 ppm) and Pusa Shukti (3.582 ppm). At 95 DAT, a significant increase in GA concentration was observed in Pusa Ashwini (3.405 ppm) and Pusa Sharad (4.730 ppm), while Pusa Shukti (3.885 ppm) and PSB Kt-25 (3.205 ppm) had slight increases.
The HPLC chromatograms for one replication are presented in S3 Fig.
Changes in GA3 content over 35 DAT
In cauliflower, 35-day-old plants correspond to the young stage following which crucial developmental transitions occur; hence, this stage was taken as the base line for calculating the relative change in GA3 content. In Pusa Ashwini, the highest reduction (-26.81%) in GA3 content compared to 35 DAT occurred at 55 DAT (Fig 4B). For Pusa Sharad, the GA3 content was higher compared to that at 35 DAT at all time intervals, with the largest increase at 55 DAT (41.88%), followed by 95 DAT (40.29%). Pusa Shukti had a GA3 content lower than the base point at all time intervals, with the greatest reduction (-16.83%) at 55 DAT. Like in Pusa Shukti, in PSB Kt-25, the GA3 content remained lower than the base point across all time intervals. The largest reduction in PSB Kt-25 was observed at 75 DAT (-29.72%), which was quite close to the value at 95 DAT (-28.90%).
Correlation analysis
The correlation analysis between the GA3 content of developmental transitions and phenological traits is presented in Fig 5A. It shows that the GA3 content in leaves at full curd stage (FC) was strongly correlated with GA3 content in leaves at the young stage (r = 0.77), adult stage (r = 0.84), and curd initiation stage (r = 0.96). However, a negative correlation was observed between the GA3 content in the curd portion and the GA3 content in the leaf portion from all stages. Furthermore, GA3 content at the young stage, adult stage, curd initiation stage, and full curd stage was strongly correlated to days to curd initiation (DCI) stage, days to full curd (DFC) stage, and days to bolting stage (DBS). Similarly, GA3 content in leaf and stalk portions at the bolting stage showed a strong positive correlation. Moreover, GA3 content at time points at 55 DAT, 75 DAT, and 95 DAT was strongly correlated with each other, pointing to a connection between temporal changes (Fig 5B).
(A) Correlation plots for GA3 content at developmental transitions and phenological observations. (B) Correlation plots for GA3 content at time points. SS - Seedling Stage, YS - Young Stage, AS - Adult Stage, CI - Curd Initiation, FC - Full Curd, BS - Bolting Stage, DYS - Days to Young Stage, DAS - Days to Adult Stage, DCI - Days to Curd Initiation, DFC - Days to Full Curd, DBS - Days to Bolting Stage, DAT - Days After Transplanting.
Discussion
Phytohormones encompass a diverse range of chemical compounds that, at micromolar or lower concentrations, play pivotal roles in regulating various aspects of plant growth and development, starting from seed germination and extending to seed maturation and seed dormancy. Gibberellins (GAs) are one of five classical hormones, along with auxins, cytokinins, abscisic acid, and ethylene [27]. Research on gibberellins has advanced for more than a century since their initial discovery [28]. GA3 was the first gibberellic acid to be structurally characterized [20] and has been extensively studied for its influence on plant physiology. In contrast to the extensively studied effects of exogenous GA3, the changes in endogenous GA3 during plant development, especially in cauliflower, are not well understood. To discern the changes in endogenous GA3 during plant development, six crucial developmental transitions and four varieties representing each maturity group of cauliflower were selected for the study.
There is cross-talk between abscisic acid (ABA) and GA during germination [29]. GA promotes seed germination, while abscisic acid (ABA) plays a role in initiating and sustaining seed dormancy [30]. Research has shown that both endogenous GAs and the external application of GA3 contribute to the promotion of seed germination in many crop plants [31]. External application of GA3 was also shown to increase seedling height [31]. Gibberellin biosynthesis in developing seeds causes the accumulation of GA precursors and/or bioactive GAs, such as GA3, GA1, and GA4 [29]. All the tested genotypes had higher concentrations of endogenous GA3 at the seedling stage, with a greater concentration in the late-genotype PSB Kt-25 (5.088 ppm), followed by the early cultivar Pusa Ashwini (4.115 ppm), and relatively low concentrations in Pusa Sharad (3.317 ppm) and Pusa Shukti (3.707 ppm). At the molecular level, GA promotes seed germination by enhancing the proteasome-mediated degradation of RGA-LIKE2 (RGL2), a DELLA repressor that inhibits germination, while ABA induces the expression of ABA-INSENSITIVE5 (ABI5), a leucine zipper transcription factor that acts as a repressor of seed germination [32].
In Arabidopsis, GAs are known to play a role in the transition from the juvenile to the adult phase, as evident from the increased levels of endogenous GAs during this transition and the induced differentiation of trichomes on the abaxial surface of leaves through GA treatments [33]. A significant increase in internal GA3 content was observed in Pusa Ashwini (2.791 ppm) and Pusa Shukti (4.443 ppm) from the young to the adult phase. In contrast, Pusa Sharad (mid-early) exhibited no change, and PSB Kt-25 (late) had a significant reduction in internal GA3 concentration from the young to the adult stage. Pertaining to time points, Pusa Sharad had a notable increase in endogenous GA3 content from 3.372 ppm at 35 DAT (young stage) to 4.784 ppm at 55 DAT (adult stage).
GAs are renowned for accelerating the vegetative-to-reproductive transition. Endogenous GAs have been shown to be a determinant in the initiation of curd formation in cauliflower [34–36], although the specific GAs involved were not identified. The exogenous application of GA3 [6,11,18,37,38] and GA4+7 [6,39] has been found to induce earliness in cauliflower and broccoli by reducing the duration of the vegetative phase and the number of vegetative nodes [6]. At the molecular level, however, exogenous GAs did not upregulate the expression of the meristem-identity genes BoAP1-a and BoAP1-c, which are associated with reproductive transition in cauliflower [6]. In the present study, at the curd initiation stage, all varieties exhibited elevated GA3 levels, with Pusa Shukti at 4.184 ppm, PSB Kt-25 at 3.771 ppm, Pusa Sharad at 3.596 ppm, and Pusa Ashwini at 3.339 ppm. Pusa Ashwini and PSB Kt-25 had significant increases in GA3 content compared with the adult stage. As far as different time-points are concerned, at 75 DAT, a higher GA3 content was observed for Pusa Ashwini (2.748 ppm) and Pusa Sharad (4.359 ppm), while at 95 DAT, Pusa Shukti exhibited increased GA3 levels (3.885 ppm). These instances coincided with the curd initiation stage in Pusa Ashwini and near-curd initiation in both Pusa Sharad and Pusa Shukti. The endogenous GA3 concentration in leaf tissues decreased significantly when all the genotypes transitioned from the curd initiation stage to the full curd stage. Similar results have been observed with exogenous GAs in previous studies. The application of exogenous GA3 and GA4+7 during the adult vegetative stage promoted curd initiation, whereas their application to the inflorescence meristem (curd) did not induce further developmental transition [6] or alter the quality of the curd [23]. In broccoli, as well, the exogenous GAs had no effect on the arrested stage, with the developed flower buds remaining unopened [6]. These findings probably suggest the role of other GAs or plant hormones in the post-inflorescence meristem (post-curd) transition.
In the present investigation, a higher concentration of GA3 was detected in the curd tissues of the genotypes of all maturity groups, ranging from 2.765 ppm in Pusa Shukti (mid-late) to 4.620 ppm in Pusa Sharad (mid-early), suggesting the significance of GA3 in curd development, which was higher than that previously reported [6]. They identified GAs, namely GA8, GA29, GA1, GA20, GA19, and GA3 in the curd tissues of cauliflower through gas chromatography-mass spectrometry (GC-MS), reporting that GA29 (0.167 ppm) was the most prominent, followed by GA19 (0.004 ppm).
GA plays a pivotal role in promoting plant bolting and flowering [12]. Substantial evidence, including mutant analysis, studies on hormone interactions, and quantitative genetics, strongly supports the role of GAs in stem elongation [40]. GAs are reported as the causal factors for stalk elongation in cabbage and broccoli [6,21]. The application of exogenous GAs has been shown to elongate bolting stalks in cauliflower (GA3 [6,11,18], GA4+7 [6]), broccoli (GA3 [6,16]), cabbage (GA1 and GA4 [21]), and Chinese cabbage [12]. In cauliflower, a study involving the estimation of endogenous GA1 reported that vernalization-mediated increases in GA₁ levels in leaf and stalk tissues play a causal role in stalk elongation [11]. However, in the present study, the endogenous GA3 content was significantly higher at the bolting stage in both the leaf and stalk tissues of all four genotypes without vernalization treatment. Furthermore, correlation analysis indicated a strong positive correlation between GA3 content in leaf and stalk tissues at the bolting stage, as well as between GA3 content in the leaf at the bolting stage and the days to bolting stage. Additionally, stalk length also showed a positive correlation with GA3 content at the bolting stage in both leaf and stalk tissues. When analyzed at different time points after transplanting, Pusa Ashwini exhibited a significant increase in endogenous GA3 at 95 DAT, coinciding with its bolting stage. These findings suggest that endogenous GA₃ plays a crucial role in regulating the time to bolting and the associated stalk elongation in cauliflower without vernalization.
The GA3 content at the full curd stage was strongly correlated with the GA3 content at the young stage, adult stage, and curd initiation. Furthermore, a strong positive correlation of the duration to curd initiation, curd maturity, and bolting stage with GA3 concentration at the young stage, adult stage, curd initiation, and full curd stage indicates a crucial role of GA3 in regulating these developmental transitions [18,22,41]. However, the GA3 content at the bolting stage in both leaf and stalk tissues was negatively correlated with the GA3 content in curd tissues.
The elevated levels of endogenous GA3 during the seedling stage, curd initiation stage, and bolting stage suggest the potential associations of internal GA3 with seedling elongation, curd induction, and stalk elongation associated with bolting. The time-point results of GA3 content corresponded with the stage of development. To determine the role of phytohormones in developmental transitions, it is recommended that the levels of other endogenous GAs from both the 13-hydroxylation pathway and the non-13-hydroxylation pathway be explored, as it remains unclear whether both pathways are active in cauliflower [6]. Additionally, examining their interactions with other plant hormones is important, as hormonal pathways are interconnected through a complex network of interactions and feedback circuits, which collectively determine the overall effects of individual hormones [42]. To uncover the molecular mechanisms underlying the influence of GAs on developmental stages, expression studies of genes related to the GA pathway and the ambient temperature pathway are also imperative.
Conclusion
This investigation confirmed the impact of developmental transitions on changes in endogenous GA3, indicating the putative role of GA3 in governing these transitions. A higher GA3 content at the bolting stage suggested an associated role of GA3 in stalk elongation during bolting. GA3 was also detected at a higher concentration in curd and stalk tissues at the full curd and bolting stages, respectively. Fluctuations at different time points followed the corresponding developmental stages of the plant. The maturity groups exhibited significant variation in GA3 content during both developmental transitions and time points.
Supporting Information
S1 Fig. Step-by-step protocol of sample preparation for GA3 content analysis by HPLC.
https://doi.org/10.1371/journal.pone.0321599.s001
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S2 Fig. Standard curve (Concentration vs Area) of GA3.
https://doi.org/10.1371/journal.pone.0321599.s002
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S3 Fig. HPLC chromatograms (A) HPLC chromatograms of GA3 content at developmental transitions and (B) HPLC chromatograms of GA3 content at time points.
A peak of GA3 was observed at 5.4–5.6.
https://doi.org/10.1371/journal.pone.0321599.s003
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S1 Table. Replication-wise days to developmental transitions and stalk length during 30 August 2022 sowing.
https://doi.org/10.1371/journal.pone.0321599.s004
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S2 Table. Replication-wise days to developmental transitions during 30 October 2022 sowing.
https://doi.org/10.1371/journal.pone.0321599.s005
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S3 Table. Replication-wise GA3 content (ppm) in leaf portion of cauliflower during six developmental transitions (sowing date: 30 August 2022).
https://doi.org/10.1371/journal.pone.0321599.s006
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S4 Table. Replication-wise GA3 content (ppm) in curd and stalk portions of cauliflower (sowing date: 30 August 2022).
https://doi.org/10.1371/journal.pone.0321599.s007
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S5 Table. Replication-wise GA3 content (ppm) in leaf portion of cauliflower at four time points (sowing date: 30 October 2022).
https://doi.org/10.1371/journal.pone.0321599.s008
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Acknowledgments
The senior author thanks the Director, ICAR-IARI, New Delhi, for providing research facilities.
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