Effects of water-deficit stress and foliar-applied gibberellic acid (GA3) on ‘Washington’ navel orange (Citrus sinensis) floral gene expression and inflorescence number were quantified. Trees subjected to 8 weeks of water-deficit stress [average stem water potential (SWP) −2.86 MPa] followed by 3 weeks of re-irrigation (SWP recovered to > −1.00 MPa) produced more inflorescences in week 11 than trees well-irrigated (SWP > −1.00 MPa) for the full 11 weeks (P < 0.001). After 8 weeks of water-deficit stress, bud expression of flowering locus t (FT), suppressor of overexpression of constans1 (SOC1), leafy (LFY), apetala1 (AP1), apetala2 (AP2), sepallata1 (SEP1), pistillata (PI), and agamous (AG) increased during the re-irrigation period (weeks 9 and 10), but only AP1, AP2, SEP1, PI, and AG expression increased to levels significantly greater than that of well-irrigated trees. Foliar-applied GA3 (50 mg·L−1) in weeks 2 through 8 of the water-deficit stress treatment did not reduce bud FT, SOC1, or LFY expression, but prevented the upregulation AP1, AP2, SEP1, PI, and AG expression that occurred during re-irrigation in water-deficit stressed trees not treated with GA3. Applications of GA3 to water-deficit stressed trees reduced inflorescence number 95% compared with stressed trees without GA3. Thus, GA3 inhibited citrus (Citrus sp.) floral development in response to water-deficit stress through downregulating AP1 and AP2 expression, which likely led to the failed activation of the downstream floral organ identity genes. The results reported herein suggest that bud determinacy and subsequent floral development in response to water-deficit stress in ‘Washington’ navel orange are controlled by AP1 and AP2 transcript levels, which regulate downstream floral organ identity gene activity and the effect of GA3 on citrus flower formation. The water-deficit stress floral-induction pathway provides an alternative to low-temperature induction that increases the potential for successful flowering in citrus trees grown in areas experiencing warmer, drier winters due to global climate change.
Effects of water-deficit stress and foliar-applied gibberellic acid (GA3) on ‘Washington’ navel orange (Citrus sinensis) floral gene expression and inflorescence number were quantified. Trees subjected to 8 weeks of water-deficit stress [average stem water potential (SWP) −2.86 MPa] followed by 3 weeks of re-irrigation (SWP recovered to > −1.00 MPa) produced more inflorescences in week 11 than trees well-irrigated (SWP > −1.00 MPa) for the full 11 weeks (P < 0.001). After 8 weeks of water-deficit stress, bud expression of flowering locus t (FT), suppressor of overexpression of constans1 (SOC1), leafy (LFY), apetala1 (AP1), apetala2 (AP2), sepallata1 (SEP1), pistillata (PI), and agamous (AG) increased during the re-irrigation period (weeks 9 and 10), but only AP1, AP2, SEP1, PI, and AG expression increased to levels significantly greater than that of well-irrigated trees. Foliar-applied GA3 (50 mg·L−1) in weeks 2 through 8 of the water-deficit stress treatment did not reduce bud FT, SOC1, or LFY expression, but prevented the upregulation AP1, AP2, SEP1, PI, and AG expression that occurred during re-irrigation in water-deficit stressed trees not treated with GA3. Applications of GA3 to water-deficit stressed trees reduced inflorescence number 95% compared with stressed trees without GA3. Thus, GA3 inhibited citrus (Citrus sp.) floral development in response to water-deficit stress through downregulating AP1 and AP2 expression, which likely led to the failed activation of the downstream floral organ identity genes. The results reported herein suggest that bud determinacy and subsequent floral development in response to water-deficit stress in ‘Washington’ navel orange are controlled by AP1 and AP2 transcript levels, which regulate downstream floral organ identity gene activity and the effect of GA3 on citrus flower formation. The water-deficit stress floral-induction pathway provides an alternative to low-temperature induction that increases the potential for successful flowering in citrus trees grown in areas experiencing warmer, drier winters due to global climate change.
Water-deficit stress, as well as low-temperature stress, is documented to induce flowering in citrus [Citrus sp. (Barbera et al., 1981, 1985; Chica and Albrigo, 2013a, 2013b; Li et al., 2017; Lovatt et al., 1988a, 1988b; Moss, 1969; Nakajima et al., 1992; Southwick and Davenport, 1986; Tang and Lovatt, 2019)]. The water-deficit floral-induction pathway in citrus is of particular interest because drought is increasingly pervasive due to climate change (Easterling et al., 2000), and drought stress can be regulated in irrigated orchards. With additional knowledge, it might be possible to improve irrigation practices, such as deficit irrigation, to limit the negative effects of drought while deriving a benefit from water deficit that increases flowering and yield in growing areas where winter low temperatures have become insufficient for flower induction due to climate change. Water-deficit stress substitutes for low temperature in citrus-growing regions near the equator that have distinct wet and dry seasons (Reuther, 1973) and is used to supplement low temperature to increase flowering in the tropical-subtropical citrus-producing areas of Florida and Brazil (Albrigo et al., 2002, 2006). In addition, water-deficit stress-induced flowering is the basis of the ancient technique of forzatura di limone developed in Sicily to produce summer (verdelli) lemons [Citrus limon (Barbera et al., 1981, 1985)]. The technique “forces” trees to flower by imposing 8 weeks of water-deficit stress during the hot summer months. At the end of the stress period, trees are re-irrigated and flower within 3 to 4 weeks. The “forzatura” crop is harvested the following summer, when lemons are in demand by consumers. For both ‘Frost Lisbon’ lemon (Lovatt et al., 1988a, 1988b) and ‘Tahiti’ lime [Citrus latifolia (Southwick and Davenport, 1986)], inflorescence and flower numbers increased in parallel with the increasing severity or duration of the water deficit imposed, providing evidence of a quantitative effect of water-deficit stress on citrus flowering.
Despite the application of water deficit in commercial citrus production to augment spring bloom (Albrigo et al., 2006) or to induce out-of-season flowering (Maranto and Hake, 1985), research investigating the role of water deficit in regulating floral development at the level of gene transcription is limited (Chica and Albrigo, 2013b; Li et al., 2017). Imposing 6 weeks of moderate water-deficit stress (SWP −2.00 MPa) to induce flowering in ‘Washington’ navel orange (C. sinensis) provided evidence that leaf expression of FT increased during the stress period and decreased to pre-stress levels on re-irrigation, whereas bud expression of SOC1, LFY, and AP1 increased only after the trees were re-irrigated; bud expression of FT was not quantified (Chica and Albrigo, 2013b). In a second experiment with 2-year-old ‘Femminello’ lemon trees on trifoliate orange (Poncirus trifoliata) rootstock that flowered after only 2 weeks of minimal water-deficit stress (−1.5 MPa) vs. well-watered control trees (−1.0 MPa), which flowered poorly, leaf FT transcripts increased during water-deficit stress and decreased following re-irrigation (Li et al., 2017). Based on RNA sequencing results, Li et al. (2017) hypothesized that activation of a leaf FT contributed to the upregulation of AP1 in buds, either directly or indirectly through SOC1, and ultimately resulted in the activation of the downstream floral organ identity genes. Roles for bud FT and LFY were not identified (Li et al., 2017). Whereas the results of these two studies suggest that floral induction is regulated by a leaf FT in response to water deficit, the role of bud FT and downstream genetic control of bud determinacy and floral organ identity in response to drought have yet to be delineated. Furthermore, it is currently not known whether water-deficit stress-induced flowering in citrus proceeds by the same floral development pathway as low-temperature–induced flowering or entails unique regulatory events. Having this information might prove valuable for improving the use of water deficit alone or in conjunction with low temperature to increase citrus flowering and yield.
Regulation of citrus floral development at the level of gene transcription is also of scientific interest and practical importance because, in contrast to Arabidopsis thaliana (Wilson et al., 1992), GA3 inhibits citrus flowering (Guardiola et al., 1982; Lord and Eckard, 1987; Tang and Lovatt, 2019). Exogenous applications of GA3 to citrus result in continued vegetative development of the shoot apical meristem (SAM) if applied before the SAM is determined (irreversibly committed to floral development) (Lord and Eckard, 1987). As a result, care must be taken to properly time citrus cultural management practices involving foliar-applied GA3 to avoid negative effects on spring bloom (Lovatt and Coggins, 2019; Tang et al., 2021; Vashisth et al., 2020). It is important to note that thus far, the inhibitory effect of GA3 on citrus floral development has been demonstrated only under conditions in which low temperature was the stimulus for flowering and that results observed at the level of gene transcription in these experiments have been variable. For example, in the Northern Hemisphere, flowering was reduced in ‘Salustiana’ sweet orange (C. sinensis) by a single spray of GA3 (40 mg·L−1) in early December, which reduced FT expression in leaves and FT and AP1 expression in buds, with no effect on bud expression of SOC1 or LFY (Goldberg-Moeller et al., 2013). In contrast, for ‘Orri’ mandarin (Citrus reticulata × Citrus temple), four foliar applications of GA3 (150 mg·L−1) made every 2 weeks starting in mid-November through the end of December reduced bud expression of FT, but had no effect on bud SOC1 or AP1 expression, despite reducing flowering (Muñoz-Fambuena et al., 2012a). In a third experiment, seven weekly applications of GA3 (50 mg·L−1) to ‘Washington’ navel orange trees during weeks 2 through 8 of an 8-week low-temperature floral-induction treatment had no effect on bud expression of FT, SOC1, or LFY, but reduced bud expression of AP1 and AP2, totally repressed SEP1, PI, and AG expression, and reduced inflorescence number to that of control trees under warm-temperature well-irrigated conditions (Tang and Lovatt, 2019). Taken together, the results published to date fail to establish whether inhibition of citrus floral development by GA3 is mediated at FT and/or by AP1 and AP2.
Water-deficit stress and well-irrigated treatments provide a second, independent system from low and warm temperatures for promoting and preventing flowering in citrus, respectively, that has been underused in research investigating the roles played by specific genes in regulating floral initiation, floral meristem determinacy, and floral organ specification. Water-deficit stress also provides an additional tool for identifying the genes mediating the inhibitory effect of GA3 on citrus floral development. Whether the effects of water deficit or GA3 on citrus flowering are initiated in the leaf or in the bud, each treatment must result in a sequence of events that takes place in the bud to promote or inhibit flowering.
Herein, we report the results of comparative analyses of the temporal patterns of expression of core genes demonstrated to function in classic networks regulating floral development across plant species (Benlloch et al., 2007), and likely regulating floral timing [induction (FT, SOC1, and LFY)], floral meristem identity [determinacy (LFY and AP1)], and floral organ identity [flower formation (AP2, SEP1, PI, and AG)] in buds of ‘Washington’ navel orange trees in response to water-deficit stress conditions known to promote significant flowering and well-irrigated conditions known to sustain vegetative shoot growth (Lovatt et al., 1988a; Southwick and Davenport, 1986). This research was undertaken with the following objectives: 1) to profile the expression patterns of key genes regulating floral development from induction to floral organ specification in citrus buds in response to water-deficit stress; 2) to identify the genes conferring floral meristem determinacy when water deficit is the stimulus for flowering; and 3) using water-deficit stress as the positive flowering control, to identify the genes mediating the inhibitory effect of GA3 on citrus flowering. The developmental fate of buds (floral, vegetative, or quiescent) on all trees was determined. To the authors’ knowledge, this study is the first to use water deficit to increase floral intensity to quantify the inhibitory effect of GA3 on floral gene expression to identify the genes mediating the inhibition of flowering. Thus, this research is the first to quantify changes in citrus AP2, SEP1, PI, and AG transcript levels in relation to successful and unsuccessful flowering in response to water-deficit stress, with and without foliar-applied GA3, and well-irrigated conditions, respectively. The results provide the first evidence in citrus buds of the temporal relationship between changes in both AP1 and AP2 transcript accumulation with the upstream expression of FT and the downstream activation of the floral organ identity genes when flower formation was successful under water-deficit stress.
Five-year-old mature ‘Washington’ navel orange scions on ‘Carrizo’ citrange rootstocks (C. sinensis × P. trifoliata) grown in 56-L pots containing steam-sterilized soils composed of 50% plaster sand, 25% bark, and 25% peatmoss [v/v (Soil Mix I; University of California, Riverside Agriculture Operations, Riverside, CA)] were used in this research. The research used a complete randomized design with four ‘Washington’ navel orange trees (replications) per treatment and three treatments: (i) well-irrigated (negative control) trees, for which SWP was maintained at approximately −1.00 MPa for the 11 weeks of the experiment by daily irrigation; (ii) water-deficit stress (positive control) trees, for which SWP was maintained at less than or equal to −2.40 MPa by deficit irrigation for 8 weeks (day 0 to day 55) (Southwick and Davenport, 1986), followed by 3 weeks of re-irrigation starting on day 55 (with SWP recovering to −1.00 MPa by day 60); and (iii) water-deficit stress trees (subjected to the same stress and re-irrigation conditions described for treatment 2) sprayed weekly with 50 mg·L−1 GA3 (ProGibb 40%; Valent BioSciences, Libertyville, IL), containing 0.01% nonionic surfactant (Silwet L77; Helena Chemical, Collierville, TN), in weeks 2 through 8 (seven applications) (Fig. 1). Applications of GA3 were made in the morning to the entire tree to runoff to give full canopy coverage using a hand sprayer. The SWP values reported herein were measured according to McCutchan and Shackel (1992). For all trees in all treatments, the evening before the day of SWP measurement, three leaves per tree were randomly selected and each leaf was covered with a plastic zip lock bag and then wrapped with aluminum foil to prevent exposure to light. Between 0800 and 1000 hr the following day, each leaf was detached and SWP was determined, using a pressure chamber (PMS Instrument Company, Albany, OR). Midmorning SWP measured in this manner gives maximum SWP and is predictive of midday (lowest) SWP; both midmorning and midday SWP under and overestimate SWP by 0.3 MPa (Fulton et al., 2001). Thus, a midmorning SWP of less than −2.40 ensured that the trees were stressed. This process was repeated every 2 to 3 d during the 8-week stress period and every 5 d during the 3-week re-irrigation period, a sampling strategy that proved effective in a prior experiment. SWP for the well-watered control trees was determined on the same schedule. Note that day −6 refers to the day irrigation was withheld and day 0 refers to the first day that all water-deficit stress-treated trees reached a SWP less than −2.40 MPa, at which time the experiment was initiated (Fig. 2). For both sets of water-deficit stressed trees, the stress was inadvertently interrupted on days 14 and 15 by overwatering. On day 55, the end of water-deficit stress treatment, both sets of water-deficit stress-treated trees were re-irrigated, and by day 60, tree SWP had recovered to a nonstress level (> −1.00 MPa). After the two sets of trees subjected to water-deficit stress were re-irrigated, they were maintained under well-irrigated conditions through flowering in week 11 (a period of 3 weeks).
All trees used in this research had been maintained under well-irrigated conditions in a temperature/humidity-controlled glasshouse located in the University of California, Riverside, CA (lat. 33°58′N, long. 117°19′W), with supplemental lighting of high-pressure sodium lamps to maintain a 16-h day (500 μmol·m−2·s−1 of the PAR spectrum) for the 5 months before the start of the experiment in September, at the end of the second flush of vegetative shoot growth. All fruit were removed from the trees during this period to eliminate a potential negative effect on floral gene expression and floral intensity (Muñoz-Fambuena et al., 2012a). With the exception of irrigation and GA3 application, all trees were treated the same, including temperature [24/19 °C (day/night)], daylength and light intensity [16/8 h (day/night)], fertilization (12N–1.7P–6.6K, slow-release, water-soluble fertilizer applied in the beginning of the experiment), and relative humidity (≈80%).
The distal five buds from 15 nonbearing shoots per tree were collected between 1000 and 1020 hr on each sampling day at weeks 2, 4, 6, 8, 9, and 10 from each of the four trees (four replications) per treatment, with the exception that sample collection for the GA3-treated trees was delayed until 2 weeks after the first GA3 application. Collected buds, on shoots longer than five nodes with leaves attached, were placed between moistened paper towels in a plastic bag and placed in a cooler box for immediate transport to the laboratory (≈5 min). Leaves were removed and the five distal buds on each shoot were quickly frozen in liquid nitrogen and stored at −80 °C until analyzed. Total RNA was extracted from bud tissue, previously ground in liquid nitrogen, using Isolate Plant RNA Mini Kit (Bioline USA Inc., Taunton, MA) with quality and quantity of RNA evaluated by spectrophotometry using a spectrophotometer (NanoDrop 2000; Thermo Scientific, Waltham, MA) and automated electrophoresis (2100 Bioanalyzer; Agilent Technologies, Santa Clara, CA). For complementary DNA (cDNA) synthesis, 1 μg total RNA was first treated with DNase (RQ1 RNase-Free DNase; Promega, Madison, WI) to eliminate potential DNA contamination and used in first-strand synthesis using a cDNA synthesis kit with oligo (dT) primer (Tetro cDNA Synthesis Kit, Bioline USA Inc.) in a 30-μL reaction according to the manufacture’s protocol.
The sequences of target genes, including FT, SOC1, LFY, AP1, AP2, SEP1, PI, and AG in citrus were obtained from GenBank and Reference Sequence databases of the National Center for Biotechnology Information (NCBI, Bethesda, MD). These genes were selected based on their demonstrated functional equivalence in their respective A. thaliana mutants and the fact that their expression was significantly correlated with floral intensity in response to low temperature in citrus (Nishikawa et al., 2007, 2009; Pillitteri et al., 2004; Shalom et al., 2012; Song et al., 2010; Tan and Swain, 2007; Tang and Lovatt, 2019). Gene-specific primers were designed using the web-based PrimerQuest program (Integrated DNA Technology, San Diego, CA). Annealing temperature and concentration for each primer set were optimized to the efficiency within the range of 90% to 110%. The sequences and the product sizes of the primer pairs used in this study as well as the BLAST (NCBI) results of polymerase chain reaction (PCR) product sequence vs. target sequence of each gene of interest are listed in Table 1.
Primers for the target and reference genes used in the quantitative real-time polymerase chain reactions (PCR).
Quantitative real-time PCR (qPCR) was carried out using a real-time detection system (CFX96 Touch; Bio-Rad Laboratories, Hercules, CA) in a 15-μL reaction volume containing 1.2 μL cDNA (about 40 ng of input RNA), 0.6 μL gene-specific forward and reverse primer mix (10 ng), 7.5 μL SYBR green reagent mix [SensiMix SYBR Fluorescein (2X); Bioline USA Inc.], and 5.7 μL PCR-grade water. Each reaction was run at 95 °C for 10 min followed by 40 cycles of 95 °C for 10 s and 60 °C for 1 min. Melt-curve analysis ranging from 60 to 95 °C was performed at the end of each qPCR run to confirm that nonspecific products were not formed. Using quantification cycle (Cq) values less than 35 obtained from qPCR, relative levels of expression (fold change) of the genes of interest were calculated using the Pfaffl method (Pfaffl, 2001), with beta-actin (ACT) as the reference gene (endogenous control) and ‘Washington’ navel orange flowers collected from orchard trees at spring bloom as the calibrator control (expression level of 1). Note that the expression of LFY in the ‘Washington’ navel orange flower was low and resulted in the high expression values reported in Fig. 3C. To verify the results, the data were analyzed using buds collected on day −6 (1 d before irrigation was withdrawn) as the control (expression level of 1). This reduced the expression levels for LFY with no substantive change in the results. The selection of ACT as the primary reference gene was based on its stability in qPCR analysis across citrus genotypes and tissues (Yan et al., 2012). The data obtained using ACT as the primary reference gene were compared with data obtained using elongation factor 1-alpha (EF1-α) (Nishikawa et al., 2009) as a second reference gene. No substantial differences in the results or interpretation of the data reported herein were found (data not shown). Gene expression data for each treatment and sample date were the mean of four biological replicates; each biological replicate was the mean of three qPCR technical replicates.
Maximum bloom occurred at week 11 for trees exposed to 8 weeks of water-deficit stress and re-irrigated for 3 weeks. At this time, the fate of the distal five buds on each of the 15 nonbearing shoots randomly selected from 100 to 120 shoots per tree was determined as the number of leafless (one to many flowers with no leaves), leafy (one to many flowers with one to many leaves), and total inflorescences (sum of leafless plus leafy inflorescences), vegetative shoots, and quiescent buds (inactive, did not produce a floral or vegetative shoot within the duration of the experiment) per tree for trees in all treatments. Results for the five distal buds on the 15 shoots per tree were averaged for the four individual trees (replications) per treatment and reported as the average value per tree.
Analysis of variance (ANOVA) was used to test for treatment effects on the number of inflorescences, vegetative shoots, and quiescent buds per tree using the general linear model procedure of SAS (version 9.3; SAS Institute Inc., Cary, NC). Because buds were collected over time, treatment effects on expression levels of each gene (after square root transformation of the data to stabilize variance) on each sampling date were determined using the linear mixed model procedure with time as the repeated factor. The changes in gene expression levels over time within each treatment were also determined using the same procedure. When ANOVA testing indicated significant differences, post hoc comparisons were run using Tukey’s honestly significant difference procedure. Data were back-transformed for presentation in Figs. 3–5. When the expression level of the target gene in each of the four biological replications was below the threshold value for detection (Cq in qPCR ≥ 35), the results were reported as not detected (ND) (Figs. 3 and 5). Pearson’s product moment correlation coefficients were calculated to identify significant relationships (r > 0.5, P ≤ 0.05) between average SWP and the developmental fate of buds and between gene expression level and inflorescence number, respectively. Significant correlations were subjected to regression analyses, using the least squares method for the generalized linear model.
For ‘Washington’ navel orange trees grown under well-irrigated conditions for 11 weeks, SWP averaged −0.70 MPa and was greater than −1.00 MPa on all sampling dates except one (Fig. 2), indicating that the trees were not experiencing water-deficit stress (Hake, 1995; Li et al., 2017). For trees in the water-deficit stress treatment, SWP decreased to −2.70 MPa after irrigation had been withheld for 5 d and was subsequently maintained at less than or equal to −2.40 MPa for most of the 54-d treatment period (Fig. 2). Averaged across the 54 d of water-deficit stress, the SWP for the two sets of water-deficit stress-treated trees was not significantly different; compare −2.86 ± 0.65 MPa for stressed trees not treated with GA3 to −2.70 ± 0.62 MPa for stressed trees treated with GA3 (Table 2, Fig. 2). The results document that the trees in the two stress treatments were subjected to water deficit of equivalent severity. In addition, when re-irrigated, both sets of trees recovered from water-deficit stress within 5 d [SWP > −1.00 MPa (Fig. 2)]. Thus, differences in floral intensity and gene expression observed for the two sets of trees were solely due to the exclusion or inclusion of GA3 applications in the treatment. Importantly, both sets of trees subjected to water-deficit stress had SWPs significantly lower than those of the well-irrigated trees throughout the 54-d water-deficit period and equal to those of well-irrigated trees in weeks 9 through 11 [P < 0.001 (Fig. 2)].
Average stem water potential (SWP) during the first 8 weeks of treatment (from day 0 to day 54) and the developmental fate of buds of ‘Washington’ navel orange trees grown under (i) well-irrigated [WI (SWP > −1.00 MPa)] conditions for 11 weeks; (ii) 8 weeks of water-deficit (WD) stress (SWP ≤ −2.40 MPa) followed by 3 weeks of WI; and (iii) 8 weeks of WD stress (SWP ≤ −2.40 MPa) plus weekly foliar applications of 50 mg·L−1 gibberellic acid (GA3) in weeks 2 through 8 followed by 3 weeks of WI.
Trees that were well-irrigated for 11 weeks (negative control) produced an average of only 0.8 total inflorescence per tree (based in all cases on five buds/15 shoots per tree) (Table 2), indicating that most of the buds collected and analyzed in this research were not committed to floral development at the initiation of the experiment. In contrast, trees subjected to 8 weeks of water-deficit stress (positive control) produced 51 inflorescences per tree, a significantly greater number than that of well-irrigated trees (P < 0.001). Average SWP during the first 8 weeks of the experiment was significantly negatively correlated with the number of inflorescences produced per tree at week 11 (r = −0.97, P < 0.001) and average SWP explained 93% of the variation in inflorescence number. Leafless and leafy inflorescences comprised 57% and 43%, respectively, of the total inflorescences produced by trees subjected to 8 weeks of water-deficit stress. The number of leafless inflorescences produced by water-deficit stressed trees was numerically greater than that of well-irrigated trees (Table 2), but not significantly greater due to the variation in the number of leafless inflorescences produce by the four trees (replications) in the water-deficit stress treatment. The number of leafy inflorescences was significantly increased on trees subjected to 8 weeks of water-deficit stress compared with well-irrigated trees (P < 0.01) and was significantly negatively correlated with average SWP of individual trees (r = −0.80, P = 0.018). Water-deficit stress had no effect on the number of vegetative shoots produced per tree (Table 2).
The total number of inflorescences produced by trees subjected to 8 weeks of water-deficit stress (51/tree) was reduced 95% by weekly foliar applications of GA3 during weeks 2 through 8 to only 0.3 inflorescence per tree (P < 0.001), a number not statistically different from that of 11-week well-irrigated trees (Table 2). In contrast, GA3 treatment increased vegetative shoot number (> 2.5-fold) to 23.3 per tree compared with trees subjected to water-deficit stress but not treated with GA3 (P < 0.01). Well-irrigated trees produced even fewer vegetative shoots (0.5 vegetative shoot/tree). For the 11-week well-irrigated trees, most buds (73.8/75 buds/tree) remained quiescent (inactive, producing neither floral, nor vegetative shoots) (Table 2). Eight weeks of water-deficit stress significantly reduced the number of quiescent buds to 15.3 per tree compared with trees that were well-irrigated for 11 weeks (P < 0.001). The number of quiescent buds was significantly positively correlated with the average SWP during the 8 weeks of the water-deficit treatment (r = 0.99, P < 0.001), with average SWP explaining 98% of the variation in the number of quiescent buds. The relationship between SWP and number of quiescent buds was largely due to the positive effect of water-deficit stress on inflorescence development; the number of quiescent buds was significantly negatively correlated with the total number of inflorescences per tree across all three treatments (r = −0.96, P < 0.001), but not with vegetative shoot number (r = −0.42, P = 0.299).
Transcripts of FT, SOC1, and LFY were detected in buds of trees that were well-irrigated for 11 weeks at the beginning of the experiment, and fluctuated significantly over time (P = 0.001, P < 0.001, and P = 0.004, respectively) (Fig. 3A–C). For trees subjected to 8 weeks of water deficit, bud FT, SOC1, and LFY expression levels during the stress period were not significantly different from those of well-irrigated control trees, except in week 8 when transcripts of FT and LFY were not detected and transcripts of SOC1 were reduced 50% in well-irrigated trees, whereas they were continuously expressed in buds of water-deficit stressed trees. After the stressed trees were re-irrigated and SWP recovered to greater than −1.00 MPa, the expression of the three floral timing genes increased significantly in week 9 (FT and LFY) or week 10 (SOC1) compared with week 8 (P < 0.001 for all). However, in comparison with 11-week well-irrigated trees, only SOC1 expression was greater in stressed trees and only in week 10 (P = 0.024); there was no significant difference in FT and LFY expression between the two treatments during the re-irrigation period. It should be noted that FT, SOC1, and LFY transcripts were present in buds of all ‘Washington’ navel orange trees before withdrawing irrigation, despite the trees being maintained for the previous 5 months under well-irrigated warm-temperature conditions that did not result in flowering. The developmental significance of the apparent synchronous decline in bud FT and LFY transcript levels to below the limit of detection (ND) and 50% decrease in SOC1 transcript levels observed in week 8 for well-irrigated trees is unclear (Fig. 3A–C).
For trees receiving seven weekly applications of GA3 starting in week 2 of the 8-week water-deficit stress treatment, bud expression levels for FT (P = 0.046), SOC1 (P < 0.001), and LFY (P < 0.001) were significantly greater by week 4 compared with water-deficit stressed trees not receiving GA3 and well-irrigated trees (Fig. 3A–C). For weeks 6 through 10, there were no significant differences in bud expression of FT, SOC1, or LFY among the three treatments, with the exception of week 8, when the expression of all three genes in well-irrigated trees decreased dramatically as discussed previously, resulting in greater expression of FT, SOC1, or LFY in both sets of water-deficit stressed trees (Fig. 3A–C). Most noteworthy, GA3 had no inhibitory effect on FT, SOC1, or LFY expression in buds of ‘Washington’ navel orange trees subjected to 8 weeks of water-deficit stress on any sampling date, despite reducing total inflorescence number 95% (Fig. 3A–C, Table 2). Of additional importance, total inflorescence number was not significantly related to the expression level of any floral timing gene across all treatments and sampling dates.
In buds of 11-week well-irrigated trees, AP1 expression decreased significantly (72%) in week 8 compared with previous weeks (Fig. 4A). The greater transcript levels observed in weeks 2, 4, and 6 were not observed in week 9 (P = 0.015). In contrast, for trees subjected to water-deficit stress, despite a 33% decrease in expression in week 8 compared with the first 6 weeks, AP1 expression significantly increased (>2-fold) from week 8 to a maximum value in week 10, after 2 weeks of re-irrigation (P = 0.001). As a result, transcript levels of AP1 in buds of the water-deficit stressed trees were 3- and 4-fold greater than those of well-irrigated trees at weeks 9 and 10 (P < 0.001 for both weeks), respectively.
Bud AP2 expression was significantly lower in weeks 2 and 4 than week 6 but returned to levels similar to week 2 during weeks 8 through 10 for well-irrigated trees [P = 0.024 (Fig. 4B)]. For trees subjected to water-deficit stress for 8 weeks, AP2 expression did not change significantly from weeks 2 through 8, but increased after re-irrigation 7-fold from week 8 to a maximum value at week 10 (P < 0.001). Thus, with re-irrigation, trees subjected to 8 weeks of water-deficit stress exhibited significantly greater AP2 expression by the end of week 9 (P = 0.046) and week 10 (P = 0.003) than well-irrigated trees.
Seven weekly applications of GA3 to water-deficit stress-treated trees had no effect on AP1 expression during the stress period, but significantly reduced AP1 transcript levels during the re-irrigation period by 40% and 63% at the end of weeks 9 and 10 (P < 0.001), respectively, relative to water-deficit stress-treated trees not receiving GA3 (Fig. 4A). As a result, AP1 transcript levels in buds of GA3-treated trees were equal to those of the well-irrigated (negative control) trees by week 10. Similarly, application of GA3 had no effect or increased AP2 expression during the 8-week water-deficit stress period, but subsequently prevented the significant increase in AP2 transcript accumulation that occurred during the re-irrigation period in buds of water-deficit stressed trees not treated with GA3, reducing AP2 expression more than 50% by week 10 [P = 0.003 (Fig. 4B)]. Inflorescence number was significantly correlated across all treatments with the expression levels of AP1 at week 9 (r = 0.89, P < 0.001) and week 10 (r = 0.91, P < 0.001), and AP2 at week 9 (r = 0.63, P = 0.028) and week 10 (r = 0.89, P < 0.001).
Transcripts of SEP1 were not detected on any sampling date in buds of trees that were well-irrigated for 11 weeks (Fig. 5A). For water-deficit stress-treated trees, SEP1 transcripts were first detected after SWP returned to a nonstressed level (> −1.00 MPa) during the first week of the re-irrigation period that followed the 8 weeks of stress. Maximum SEP1 expression occurred at week 10 (P < 0.001). Similarly, PI transcripts were not detected in buds of well-irrigated trees for the duration of the 11-week experiment and only detected in water-deficit stressed trees after the trees were re-irrigated (Fig. 5B). For water-deficit stress-treated trees, bud PI transcript levels increased 5-fold from week 9 to a maximum value at week 10 (2 weeks after re-irrigation). Transcripts of AG were also not detected in buds of well-irrigated trees at any time during the 11-week experiment (Fig. 5C). Bud AG expression was also not detected in water-deficit stressed trees during the 8-week stress period. However, after re-irrigation, AG transcript levels increased 9-fold from week 9 to a maximum level at week 10 (2 weeks after re-irrigation).
Weekly application of GA3 to ‘Washington’ navel orange trees in weeks 2 through 8 of the 8-week water-deficit stress treatment inhibited the accumulation of SEP1, PI, and AG transcripts that occurred during the re-irrigation period in buds of trees subjected to 8 weeks of water-deficit stress but not treated with GA3 (Fig. 5A–C). Thus, SEP1, PI, and AG all failed to reach the maximum values of expression in week 10 for water-deficit stress-treated trees applied with GA3. Total inflorescence number was strongly correlated across all treatments with the expression levels of SEP1 at week 9 (r = 0.83, P = 0.003) and week 10 (r = 0.88, P < 0.001), PI at week 10 (r = 0.90, P < 0.001), and AG at week 10 (r = 0.91, P < 0.001).
In citrus, as in other woody perennial tree crops, the floral development process is protracted. Successful transition from a vegetative to reproductive SAM occurs many months before floral organogenesis and spring bloom. Environmental conditions at the time of key developmental events are critical to successful flowering and commercial tree crop productivity. Flowering must be timed to environmental conditions that favor flower formation and retention, successful syngamy, including synchrony with pollinizer bloom and pollinator activity in out-crossing species, and promote fruit set of seeded and parthenocarpic fruit, such as the ‘Washington’ navel orange (Hong and Jackson, 2015).
The results of this research are consistent with earlier reports that various drought treatments, independent of photoperiod, promoted flowering in citrus (Barbera et al., 1981, 1985; Chica and Albrigo, 2013b; Li et al., 2017; Lovatt et al., 1988a, 1988b; Moss, 1969; Nakajima et al., 1992; Southwick and Davenport, 1986) and add to the growing body of literature that water deficit (drought) is an important environmental factor inducing flowering across species (Takeno, 2016). For ‘Washington’ navel orange trees subjected to 8 weeks of water-deficit stress (average SWP −2.86 MPa), after being maintained for 5 months under well-irrigated warm-temperature conditions, most (68%) of the buds produced inflorescences, 12% produced vegetative shoots, and 20% remained quiescent. In contrast, 98% of buds on well-irrigated trees remained quiescent, only 1.0% produced inflorescences, and 0.7% produced vegetative shoots, indicating that most buds were not committed to floral development at the start of the experiment. Average SWP during the first 8 weeks of the experiment was significantly negatively correlated with the number of inflorescences produced per tree at week 11, with average SWP explaining 93% of the variation in inflorescence number.
The first results documenting that GA3 inhibits water-deficit stress-induced flowering in citrus are presented herein. When GA3 was applied during weeks 2 through 8 of the 8-week water-deficit stress treatment, inflorescence number was reduced 95%, with a concomitant 63% increase in vegetative shoot number compared with trees subjected to 8 weeks of water-deficit stress not treated with GA3. Although both sets of trees were subjected to water-deficit stress of equal severity, 69% of the buds of water-deficit stress trees treated with GA3 remained quiescent. In addition to inducing floral development, 8 weeks of water-deficit stress increased percent budbreak in week 11 to 80% compared with 69% for water-deficit stressed trees treated with GA3 and 1.6% for well-irrigated trees. Inflorescence number, but not vegetative shoot number, in the present research was strongly negatively correlated with the number of quiescent buds per tree across all treatments. Thus, for ‘Washington’ navel orange, the effect of water-deficit stress on floral induction and floral budbreak was closely related. Whether this constitutes a single response or two separate responses to water deficit is unknown. In ‘Owari’ Satsuma mandarin (Citrus unshiu), the parallel increases in floral induction and budbreak due to low temperature were demonstrated to be two distinct responses (García-Luís et al., 1992).
In light of the fact that the well-irrigated trees did not flower (<1 inflorescence/75 buds/tree), it is of interest that FT, SOC1, LFY, AP1, and AP2 (at a low level) were expressed in the buds of these trees throughout the 11-week well-irrigated treatment. The expression of SOC1, LFY, and AP1 in buds of well-watered control trees was also reported by Chica and Albrigo (2013b). For trees exposed to water deficit, FT, SOC1, and LFY transcripts accumulated in buds during the re-irrigation period following the stress treatment. However, only SOC1 was expressed at a level greater than that of the well-irrigated control trees at the end of the stress period in week 8 and 2 weeks after re-irrigation in week 10. The lack of significant difference in FT expression in buds of water-deficit stressed and well-irrigated trees is consistent with the possibility that floral development in response to water deficit in this study was also initiated through activation of a leaf FT (Chica and Albrigo, 2013b; Li et al., 2017). Li et al. (2017) proposed that an activated leaf FT might upregulate bud AP1 directly or indirectly via a bud SOC1. It is tempting to speculate the significantly greater expression of SOC1 in buds of water-deficit stressed trees at weeks 8 and 10 supports a role for SOC1 in this hypothetical model. Nonetheless, it is unlikely that bud FT, SOC1, and LFY are the key regulators of bud determinacy given the fact that GA3 applied during the water-deficit stress treatment resulted in the expression of these genes at levels equal to or greater than those of water-deficit stressed trees not treated with GA3 on all sampling dates and yet, GA3 profoundly suppressed the number of inflorescences that developed by 95%. These results compared with results obtained when low temperature was the stimulus for floral induction are consistent with the lack of effect of GA3 on SOC1 and LFY expression observed in buds of ‘Salustiana’ sweet orange (Muñoz-Fambuena et al., 2012b) and ‘Orri’ mandarin (Goldberg-Moeller et al., 2013), and FT, SOC1, and LFY expression in buds of ‘Washington’ navel orange (Tang and Lovatt, 2019). Although the roles of leaf and bud FT in citrus floral development await clarification, the results of this research add to the growing body of evidence that FT is transcribed in the citrus bud itself (Goldberg-Moeller et al., 2013; Muñoz-Fambuena et al., 2012a; Tang and Lovatt, 2019; Tang et al., 2021).
A major contribution of the current research to the elucidation of citrus flower development was the analysis of transcript levels of additional core genes downstream from AP1, including AP2, SEP1, PI, and AG. Three substantive results were obtained. First, for water-deficit stressed trees, both AP1 and AP2 were upregulated from week 8, the end of the stress period, to week 9, 1 week after re-irrigation, when tree SWP was restored to greater than −1.00 MPa, and from week 9 to week 10, 2 weeks after re-irrigation, when maximum expression for both genes occurred, resulting in significantly greater transcript levels by weeks 9 and 10 than those of 11-week well-irrigated trees. Second, the floral organ identity genes SEP1, PI, and AG were activated only in buds of water-deficit stress-treated trees after re-irrigation, in parallel with the increased expression of AP1 and AP2, reaching maximum levels in week 10. In contrast, for well-irrigated control trees, SEP1 and PI transcripts remained undetectable throughout the 11-week experiment; AG was only expressed in week 9 and at a marginal level significantly lower than trees subjected to 8 weeks of water-deficit stress. Taken together, the results suggest that AP1 and AP2 were only expressed at levels sufficiently high to upregulate the downstream floral organ identity genes SEP1, PI, and AG in buds of water-deficit stressed trees, which led to flowering, but remained too low in the buds of well-irrigated trees to upregulate floral organ specification and thus, flower development failed. Importantly, according to the ABCE model for floral organ specification, activity of both class A genes, AP1 and AP2, is necessary for sepal formation (Bowman et al., 1991; Coen and Meyerowitz, 1991; Krizek and Fletcher, 2005). Of specific relevance to this research, sepal formation was identified as the developmental marker indicating irreversible commitment to floral development (bud determinacy) in ‘Washington’ navel orange (Lord and Eckard, 1987), consistent with the roles of AP1 and AP2 in regulating citrus floral meristem determinacy in response to water-deficit stress. The third substantive result was that the upregulation of AP1 and AP2 and concomitant activation of SEP1, PI, and AG in response to water-deficit stress during weeks 1 and 2 of the re-irrigation period were inhibited by GA3 applied during the water-deficit stress period, and flower production failed. These results, taken together with the absence of a negative effect of foliar-applied GA3 on bud expression of FT, SOC1, or LFY in response to water-deficit stress, identify AP1 and AP2 as the genes mediating the inhibitory effect of GA3 on water-deficit stress-induced flowering in citrus. The results provide additional evidence that adequate levels of AP1 and AP2 expression are required for the upregulation of the floral organ identity genes and flowering in response to water-deficit stress in citrus. In all cases, expression of the floral organ identity genes downstream from AP2 paralleled the expression of AP1 and AP2 and when their activity was repressed, flowering did not occur. It should be noted that bud determinacy might also require expression of at least one or possibly all four SEP genes (Ditta et al., 2004; Pelaz et al., 2000). Across all treatments, inflorescence number was not significantly related to the expression of FT, SOC1, or LFY, but was strongly correlated with the expression of AP1, AP2, SEP1, PI, and AG in week 10 (2 weeks after re-irrigation).
Striking similarities in the expression patterns of core genes regulating floral development in ‘Washington’ navel orange trees induced to flower by water-deficit in this study and low-temperature stress in a previous study (Tang and Lovatt, 2019) were identified. With the exception of the stimulus used to induce flowering, note that all biotic attributes of the trees in both experiments (such as genotype, age, and size), as well as environmental factors (including the conditions during the 5 months preceding each experiment, the length of each stress treatment and recovery period, daylength, light intensity and quality, and humidity), and GA3 treatment were the same. Eight weeks of water deficit (SWP < −2.40 MPa followed by re-irrigation to SWP > −1.00 MPa for 3 weeks) and low temperature [15/10 °C (day/night) followed by 3 weeks at 24/19 °C (day/night)] significantly increased inflorescence number compared with control trees maintained in the well-watered, warm-temperature condition [SWP > −1.00 MPa, 24/19 °C (day/night)]. Seven GA3 applications during each stress reduced inflorescence number 95% and 96%, respectively, similar to that of control trees. Bud expression of FT, SOC1, and LFY was unaffected in a manner related to inflorescence number by either stress or GA3 treatment. In contrast, in response to both stresses, expression of AP1 and AP2 significantly increased 1 week after the trees were transferred to the nonstressed condition, followed by maximum SEP1, PI, and AG expression 1 week before flowering (week 10). Applications of GA3 prevented the upregulation of these genes, which also failed to occur in the buds of control trees; neither set of trees flowered. The results of the two separate experiments provide consistent evidence supporting the role of AP1 and AP2 in regulating citrus floral meristem determinacy and the inhibitory effect of GA3 on citrus flowering.
Based on the results of the preceding comparison, it is likely that water-deficit and low-temperature stress regulate citrus floral development through overlapping pathways, whereby AP1 and AP2 expression are necessary for bud determinacy and downstream activation of SEP1, PI, and AG for flower formation. This interpretation is consistent with the ability of water-deficit stress to augment low-temperature floral induction in citrus (Albrigo et al., 2002, 2006; Chica and Albrigo, 2013b). With each stress, significant expression of SEP1, PI, and AG only occurred after trees were transferred to the nonstressed (well-irrigated warm-temperature) condition, providing evidence of a possible failsafe mechanism to synchronize flowering with periods of available water and warmer temperature (Tang and Lovatt, 2019). Specifically, activation of AP1 and AP2 would occur under drought conditions or the low temperatures of fall and winter, but expression of AP1 and AP2 to levels sufficient to confer bud determinacy and upregulate downstream floral organ identity genes, SEP1, PI, and AG, would only occur after spring temperatures were sufficiently warm or adequate water was available, thereby preventing flower production under adverse environmental conditions.
Water-deficit stress is an effective floral-induction pathway in citrus. Results presented herein provide the first evidence demonstrating that AP1 and AP2 regulate bud determinacy in response to water-deficit stress in ‘Washington’ navel orange and upregulate the expression of the downstream floral organ identity genes, resulting in successful flowering. The results also provide the first evidence suggesting that the inhibitory effect of GA3 on water-deficit stress- induced flowering in citrus is mediated by AP1 and AP2. The presence of the water-deficit stress floral-induction pathway in citrus increases the potential for floral success of the species in nature. It might also provide growers with a tool to mitigate the negative effects of climate change and promote the positive effects of water deficit on citrus flowering and yield of commercial citrus grown in areas experiencing warmer, drier winters.
Albrigo, L.G., Valiente, J.I. & Beck, H.W. 2002 Flowering expert systeme development for a phenology based citrus decision support system
Acta Hort.584 247 254 https://doi.org/10.17660/ActaHortic.2002.584.30Albrigo, L.G., Valiente, J.I. & Van Parys de Wit, C. 2006 Influence of winter and spring weather on year-to-year citrus fruit set and yield variation in São Paulo, Brazil
Proc. Intl. Soc. Citricult. X Congr.1 263 267Barbera, G., Fatta del Bosco, G. & Lo Cascio, B. 1985 Effects of water stress on lemon summer bloom: The “forzatura” technique in the Sicilian citrus industry
Acta Hort.171 391 397 https://doi.org/10.17660/ActaHortic.1985.171.36Barbera, G., Fatta del Bosco, G. & Occorso, G. 1981 Some aspects on water stress physiology of forced lemon (
Citrus limonBurm) treesProc. Intl. Soc. Citricult. IV Congr.2 522 523Benlloch, R., Berbel, A., Serrano-Mislata, A. & Madueno, F. 2007 Floral initiation and inflorescence architecture: A comparative view
Ann. Bot.100 659 676 https://doi.org/10.1093/aob/mcm146Bowman, J.L., Smyth, D.R. & Meyerowitz, E.M. 1991 Genetic interactions among floral homeotic genes of
ArabidopsisDevelopment112 1 20 https://doi.org/10.1242/dev.112.1.1Chica, E.J. & Albrigo, L.G. 2013a Changes in
CsFTtranscript abundance at the onset of low-temperature floral induction in sweet orangeJ. Amer. Soc. Hort. Sci.13 184 189 https://doi.org/10.21273/JASHS.138.3.184Chica, E.J. & Albrigo, L.G. 2013b Expression of flower promoting genes in sweet orange during floral inductive water deficits
J. Amer. Soc. Hort. Sci.138 88 94 https://doi.org/10.21273/JASHS.138.2.88Coen, E.S. & Meyerowitz, E.M. 1991 The war of the whorls: Genetic interactions controlling flower development
Nature353 31 37 https://doi.org/10.1038/353031a0Ditta, G., Pinyopich, A., Robles, P., Pelaz, S. & Yanofsky, M.F. 2004 The
SEP4Gene ofArabidopsis thalianafunctions in floral organ and meristem identityCurr. Biol.14 1935 1940 https://doi.org/10.1016/j.cub.2004.10.028Easterling, D.R., Meehl, G.A., Parmesan, C., Changnon, S.A., Karl, T.K. & Mearns, L.O. 2000 Climate extremes: Observations, modeling, and impacts
Science289 2068 2074 https://doi.org/10.1126/science.289.5487.2068Fulton, A.R., Bucher, R., Olson, B., Schwankl, L., Gilles, C., Bertagna, N., Walton, J. & Shackel, K. 2001 Rapid equilibration of leaf and stem water potential under field conditions in almonds, walnuts and prunes
HortTechnology11 609 615 https://doi.org/10.21273/HORTTECH.11.4.609García-Luís, A., Kanduser, M., Santamarina, P. & Guardiola, J.L. 1992 Low temperature influence on flowering in
Citrus. The separation of inductive and bud dormancy releasing effectsPhysiol. Plant.86 648 652 https://doi.org/10.1111/j.1399-3054.1992.tb02182.xGoldberg-Moeller, R., Shalom, L., Shlizerman, L., Samuels, S., Zur, N., Ophir, R., Blumwald, E. & Sadka, A. 2013 Effects of gibberellin treatment during flowering induction period on global gene expression and the transcription of flowering-control genes in
CitrusbudsPlant Sci.198 46 57 https://doi.org/10.1016/j.plantsci.2012.09.012Guardiola, J.L., Monerri, C. & Agusti, M. 1982 The inhibitory effect of gibberellic acid on flowering in
CitrusPhysiol. Plant.55 2 136 142 https://doi.org/10.1111/j.1399-3054.1982.tb02276.xHake, K.D 1995 Regulation of flowering in
Citrus limonby water-deficit stress and nitrogen compounds Univ. Calif., Riverside PhD Diss. 9707046Hong, Y. & Jackson, S. 2015 Floral induction and flower formation – The role and potential applications of miRNAs
Plant Biotechnol. J.13 282 292 https://doi.org/10.1111/pbi.12340Krizek, B.A. & Fletcher, J.C. 2005 Molecular mechanisms of flower development: An armchair guide
Nat. Rev. Genet.6 688 698 https://doi.org/10.1038/nrg1675Li, J.-X., Hou, X.-J., Zhu, J., Zhou, J.-J., Huang, H.-B., Yue, J.-Q., Gao, J.-Y., Du, Y.-X., Hu, C.-X., Hu, C.-G. & Zhang, J.-Z. 2017 Identification of genes associated with lemon floral transition and flower development during floral inductive water deficits: A hypothetical model
Front. Plant Sci.8 1013 https://doi.org/10.3389/fpls.2017.01013Lord, E.M. & Eckard, K.J. 1987 Shoot development in
Citrus sinensisL. (Washington navel orange). II. Alteration of developmental fate of flowering shoots after GA3 treatmentBot. Gaz.148 17 22 https://doi.org/10.1086/337623Lovatt, C.J. & Coggins, C.W. 2019 Delaying fruit senescence with gibberellic acid (GA3)
University of California integrated pest management guidelines:Citrus. Univ. Calif. ANR Publ. 3441Lovatt, C.J., Zheng, Y. & Hake, K.D. 1988a A new look at the Kraus Kraybill hypothesis and flowering in
CitrusProc. Intl. Citrus VI Congr.1 475 483Lovatt, C.J., Zheng, Y. & Hake, K.D. 1988b Demonstration of a change in nitrogen metabolism influencing flower initiation in citrus
Isr. J. Bot.37 181 188Maranto, J. & Hake, K.D. 1985 Veredelli summer lemons: A new option for California growers
Calif. Agr.39 5 4McCutchan, H. & Shackel, K.A. 1992 Stem water potential as a sensitive indicator of water stress in prune trees (
Prunus domesticaL. cv. French)J. Amer. Soc. Hort. Sci.117 606 611 https://doi.org/10.21273/JASHS.117.4.607Moss, G.I 1969 Influence of temperature and photoperiod on flower induction and inflorescence development in sweet orange (
Citrus sinensisL. Osbeck)J. Hort. Sci.44 311 320 https://doi.org/10.1080/00221589.1969.11514314Muñoz-Fambuena, N., Mesejo, C., González-Mas, M.C., Primo-Millo, E., Agustí, M. & Iglesias, D.J. 2012a Fruit load modulates flowering-related gene expression in buds of alternate-bearing ‘Moncada’ mandarin
Ann. Bot.110 6 1109 1118 https://doi.org/10.1093/aob/mcs190Muñoz-Fambuena, N., Mesejo, C., González-Mas, M.C., Iglesias, D.J., Primo-Millo, E. & Agustí, M. 2012b Gibberellic acid reduces flowering intensity in sweet orange [
Citrus sinensis(L) Osbeck] by repressingCiFTgene expressionJ. Plant Growth Regul.31 529 536 https://doi.org/10.1007/s00344-012-9263-yNakajima, Y., Susanto, S. & Hasegawa, K. 1992 Influence of water stress in autumn on flower induction and fruiting in young pomelo trees (
Citrus grandisL Osbeck)J. Jpn. Soc. Hort. Sci.62 1 15 20 https://doi.org/10.2503/jjshs.62.15Nishikawa, F., Endo, T., Shimada, T., Fujii, H., Shimizu, T. & Omura, M. 2009 Differences in seasonal expression of flowering genes between deciduous trifoliate orange and evergreen Satsuma mandarin
Tree Physiol.29 921 926 https://doi.org/10.1093/treephys/tpp021Nishikawa, F., Endo, T., Shimada, T., Fujii, H., Shimizu, T., Omura, M. & Ikoma, Y. 2007 Increased
CiFTabundance in the stem correlates with floral induction by low temperature in Satsuma mandarin (Citrus unshiuMarc.)J. Expt. Bot.58 3915 3927 https://doi.org/10.1093/jxb/erm246Pelaz, S., Ditta, G.S., Baumann, E., Wisman, E. & Yanofsky, M.F. 2000 B and C floral organ identity functions require
SEPALLATAMADS-box genesNature405 200 203 https://doi.org/10.1038/35012103Pfaffl, M.W 2001 A new mathematical model for relative quantification in real-time RT-PCR
Nucleic Acids Res.29 2002 2007 https://doi.org/10.1093/nar/29.9.e45Pillitteri, L.J., Lovatt, C.J. & Walling, L.L. 2004 Isolation and characterization of
LEAFYandAPETALA1homologues fromCitrus sinensisL Osbeck ‘Washington’J. Amer. Soc. Hort. Sci.129 846 856 https://doi.org/10.21273/JASHS.129.6.0846Reuther, W 1973 Climate and citrus behavior 280 337 Reuther, W.
The citrus industry.2nd ed. Vol. 3 Univ. Calif. Press Berkeley, CAShalom, L., Samuels, S., Zur, N., Shlizerman, L., Zemach, H., Weissberg, M., Ophir, R., Blumwald, E. & Sadka, A. 2012 Alternate bearing in citrus: Changes in the expression of flowering control genes and in global gene expression in on-versus off-crop trees
PLoS One7 e46930 https://doi.org/10.1371/journal.pone.0046930Song, C., Jia, Q., Fang, J.G., Li, F., Wang, C. & Zhang, Z. 2010 Computational identification of citrus microRNAs and target analysis in citrus expressed sequence tags
Plant Biol.12 927 934 https://doi.org/10.1111/j.1438-8677.2009.00300.xSouthwick, S.M. & Davenport, T.L. 1986 Characterization of water stress and low temperature effects on flower induction in
CitrusPlant Physiol.81 26 29 https://doi.org/10.1104/pp.81.1.26Takeno, K 2016 Stress-induced flowering: The third category of flowering response
J. Expt. Bot.67 4925 4934 https://doi.org/10.1093/jxb/erw272Tan, F.-C. & Swain, S.M. 2007 Functional characterization of
AP3,SOC1andWUShomologues from citrus (Citrus sinensis)Physiol. Plant.131 481 495 https://doi.org/10.1111/j.1399-3054.2007.00971.xTang, L. & Lovatt, C.J. 2019 Effects of low temperature and gibberellic acid on floral gene expression and floral determinacy in ‘Washington’ navel orange (
Citrus sinensisL. Osbeck)Scientia Hort.243 92 100 https://doi.org/10.1016/j.scienta.2018.08.008Tang, L., Singh, G., Dewdney, M. & Vashisth, T. 2021 Effects of exogenous gibberellic acid in Huanglongbing-affected sweet orange trees under Florida conditions. I. Flower bud emergence and flower formation
HortScience56 1531 1541 https://doi.org/10.21273/HORTSCI16080-21Vashisth, T., Oswalt, C., Zekri, M., Alferez, F. & Burrow, J.D. 2020 2020–2021 Florida citrus production guide: Plant growth regulators Hort. Sci. Dept., Univ. Florida/IFAS Ext. CMG17
Wilson, R.N., Heckman, J.W. & Somerville, C.R. 1992 Gibberellin is required for flowering in
Arabidopsis thalianaunder short daysPlant Physiol.100 403 408 https://doi.org/10.1104/pp.100.1.403Yan, J., Yuan, F., Long, G., Qin, L. & Deng, Z. 2012 Selection of reference genes for quantitative real-time RT-PCR analysis in citrus
Mol. Biol. Rep.39 1831 1838 https://doi.org/10.1007/s11033-011-0925-9