BcABF1 Plays a Role in the Feedback Regulation of Abscisic Acid Signaling via the Direct Activation of BcPYL4 Expression in Pakchoi

Abscisic acid-responsive element-binding factor 1 (ABF1), a key transcription factor in the ABA signal transduction process, regulates the expression of downstream ABA-responsive genes and is involved in modulating plant responses to abiotic stress and developmental processes. However, there is currently limited research on the feedback regulation of ABF1 in ABA signaling. This study delves into the function of BcABF1 in Pakchoi. We observed a marked increase in BcABF1 expression in leaves upon ABA induction. The overexpression of BcABF1 not only spurred Arabidopsis growth but also augmented the levels of endogenous IAA. Furthermore, BcABF1 overexpression in Arabidopsis significantly decreased leaf water loss and enhanced the expression of genes associated with drought tolerance in the ABA pathway. Intriguingly, we found that BcABF1 can directly activate BcPYL4 expression, a critical receptor in the ABA pathway. Similar to BcABF1, the overexpression of BcPYL4 in Arabidopsis also reduces leaf water loss and promotes the expression of drought and other ABA-responsive genes. Finally, our findings suggested a novel feedback regulation mechanism within the ABA signaling pathway, wherein BcABF1 positively amplifies the ABA signal by directly binding to and activating the BcPYL4 promoter.


Introduction
Abscisic acid (ABA) is a plant hormone that plays a crucial role in resisting abiotic stress and is therefore also referred to as a 'stress hormone'.ABA mediates plant responses to environmental stresses such as drought, cold, osmotic stress, and salt stress [1][2][3][4].When plants are exposed to environmental stress, ABA rapidly accumulates, enhancing the plant's adaptability to stressful conditions [5].ABA also participates in regulating various plant developmental processes, including aging, seed germination, and root elongation [6][7][8].The core components of the ABA signaling pathway include the abscisic acid receptors PYR/PYL/RCAR family; 2C-type protein phosphatase (PP2C) subfamily; and the SnRK2 kinase subfamily [9][10][11].Under normal growth conditions, PP2C binds to SnRK2, inhibiting SnRK2 kinase activity [12].When plants are under stress, ABA binds to the PYR/PYL/RCAR receptors [13].The ABA-PYR/PYL/RCAR complex binds to PP2C, inhibiting PP2C phosphatase activity and resulting in the phosphorylation of SnRK2 [14].The phosphorylated SnRK2 phosphorylates ABRE/ABF, activating ABF proteins.Activated ABF proteins bind to the ABRE sequence in the promoter of responsive genes, thereby activating the ABA signaling pathway and enhancing the plant's tolerance to environmental stress [15,16].ABA is crucial for plant growth and development and is involved in responding to environmental stress, particularly drought stress.ABA enhances the plant's ability to clear reactive oxygen species (ROS), reducing oxidative damage and increasing drought tolerance [17,18].For example, MADS23, by promoting the transcription of ABA biosynthetic genes (NCEDs), increases endogenous ABA levels, enhancing drought tolerance in rice [19].Under drought conditions, ABA mediates stomal closure, reducing the rate of water loss in plants, which is an effective method to improve plant drought resistance [20].In tomatoes, microRNA160 participates in ABA-mediated stomal regulation by inhibiting ARF10, reducing leaf water loss, maintaining leaf water balance, and enhancing tomato adaptability to drought stress [21].
In Arabidopsis, there are nine homologs of ABF/AREB [22].Via functional gain and loss studies, ABF/AREB (ABF1, AREB1/ABF2, ABF3, and AREB2/ABF4) have been identified as critical transcription factors within the ABA signaling pathway.Exhibiting functional redundancy, they collaboratively regulate stomatal opening and are involved in mediating the plant's drought response mechanisms.Relevant studies have shown that ABRE/ABF (ABF1-4) effectively enhances the expression of ADF5 (Arabidopsis actin depolymerizing factor 5), which contributes to the remodeling of the actin cytoskeleton, thereby promoting the closure of stoma and subsequently enhancing the drought tolerance of plants [23].At the same time, the interaction between ABF (ABF1-4) protein and IDD14 (INDETERMI-NATE DOMAIN 14) enhances ABF's transcriptional activity.This process leads to the induction of stomatal closure and stimulates the expression of genes responsive to abscisic acid (ABA), thereby significantly enhancing drought tolerance in Arabidopsis.This mechanism underscores the critical role of ABA in plant adaptive responses to water stress conditions [22].The overexpression of ABF3 and ABF4 leads to stomal closure and reduced transpiration [24].In contrast, the abf3 and abf2 abf3 abf4 multiple mutants exhibit increased water loss rates under drought conditions [25,26].Additionally, ABF2 and ABF4 interact with ANAC096, participating in the ABA-mediated process of stomal closure and leaf water loss [27].In Pakchoi, four Arabidopsis ABF orthologs, BcABF1-4, have been identified [28].Research on ABFs within Pakchoi remains sparse.Within the Brassicaceae family, BnaABF2 engages in stoma closure via its interaction with the DELLA protein BnaRGA, playing a role in enhancing the drought tolerance of Brassica napus [29].The overexpression of BnaABF2 heightens the stoma's responsiveness to ABA, thereby diminishing leaf water loss [30].Additionally, BnaABF3 and BnaABF4 are subject to phosphorylation by BnaCPK5, which positively influences the drought resistance of Arabidopsis [31].The molecular mechanisms by which ABF1 controls water loss in Pakchoi leaves are largely unexplored.
In order to prevent excessive responses to ABA signaling, plants have evolved a feedback regulation mechanism for ABA signaling.This mechanism not only maintains the homeostasis of ABA signaling but also promotes adaptive responses to environmental changes.Current research has demonstrated that ABF/AREB is involved in the feedback regulation of ABA signal transduction and ABA biosynthesis processes.ABFs negatively regulate the ABA signal via feedback by promoting the expression of A-group PP2C genes [32].FYVE1, an intermediate regulatory factor in the ABA signaling pathway's negative feedback regulation process, binds to PYR/PYL/RCAR receptors and promotes their degradation [33][34][35].FYVE1 can also be phosphorylated by SnRK2, and phosphorylated FYVE1 inhibits the transcriptional activation activity of ABF4.ABF4, by directly binding to the FYVE1 promoter, regulates FYVE1 expression, participating in the negative feedback regulation of the ABA signaling pathway [36].The bZIP transcription factor ABI5 regulates seed germination via feedback regulation of the expression of PYL11 and PYL12 [37].Therefore, the in-depth study of feedback regulation in the ABA pathway is crucial for understanding its significant role in plant growth and development.Clade E Growth-Regulating 1 and 2 (EGR1/2) positively regulate plant ABA signaling by modulating the phosphorylation of SnRK2.2 [38].PYLs have been identified as crucial elements in the positive feedback modulation of ABA signaling.Via the interaction and phosphorylation of MdPYL2/12, MdCPK4 plays a significant role in enhancing ABA signal transduction [39].
Pakchoi [Brassica campestris (syn.Brassica rapa) ssp.chinensis] features a brief growth cycle, with the vegetative phase spanning 1-2 months.This vegetable is favored for its ease of cultivation, high productivity, superior traits, and nutritional richness, establishing it as a vegetable of worldwide acclaim.Nevertheless, in the cultivation process, Pakchoi is vulnerable to abiotic stresses, including salt stress [40], cold stress [41], and heat stress [42].Its relatively shallow and dispersed root system, while beneficial for efficiently absorbing surface soil nutrients and moisture, renders it particularly susceptible to drought conditions.Consequently, drought poses a significant constraint on the growth of Pakchoi.Our research findings indicate that in Pakchoi, BcABF1 can enhance the plant's drought resistance by reducing leaf water loss.By binding directly to the promoter of BcPYL4, BcABF1 regulates the ABA signaling pathway via a positive feedback loop, thereby enhancing ABA signaling and rapidly increasing the drought tolerance of Pakchoi.1a).The results indicated that all the amino acid sequences exhibited high identity and contained the conserved BRLZ domain (Figure 1a).To further understand the evolutionary relationship of ABF1, we constructed a phylogenetic tree based on the ABF1 amino acid sequences of these species (Figure 1b).The branching characteristics revealed that BcABF1 and BnABF1 are on the same evolutionary branch, indicating a close genetic relationship between B. campestris and B. napus.

Expression Analysis of BcABF1
To obtain the tissue-specific expression of BcABF1 in Pakchoi, qRT-PCR analysis was completed on the root, stem, leaf, stalk, petiole, and flower.Our findings revealed that the expression level of BcABF1 was the highest expression in the leaves, with the second highest expression observed in the flowers (Figure 2a).To advance our understanding of the BcABF1 expression pattern, we engineered a GUS reporter system under the control of the BcABF1 promoter (pBcABF1:GUS) and transformed it into Arabidopsis thaliana (Col-0), resulting in the creation of the pBcABF1:GUS/Col.GUS staining of this line revealed a notable staining intensity in the leaves, primarily concentrated in the vascular and stoma (Figure 2b).Previous research has documented diurnal rhythm variations in ABF [43].We conducted a qRT-PCR analysis to assess the diurnal variations in ABF1 expression over a 24 h period in 14 d Pakchoi seedlings (Figure 2c).This analysis revealed that the expression of BcABF1 was significantly higher during daylight hours compared to nighttime.Notably, the expression peaked at Zeitgeber Time (ZT) 8. Following this peak, there was a gradual decrease in expression.Subsequently, to explore ABF1's responsiveness to ABA, we applied 50 µM ABA to 14-day Pakchoi seedlings at ZT0.Our findings revealed that, relative to untreated Pakchoi, BcABF1's expression levels surged within the initial 8 h window posttreatment, diminished noticeably from 12 to 24 h, and eventually stabilized at an elevated level by the 24 h mark.Specifically, for post-ABA treatment, the most substantial shift in BcABF1 expression occurred at ZT8, where it was threefold higher than that in untreated plants (Figure 2c).1a).The results indicated that all the amino acid sequences exhibited high identity and contained the conserved BRLZ domain (Figure 1a).To further understand the evolutionary relationship of ABF1, we constructed a phylogenetic tree based on the ABF1 amino acid sequences of these species (Figure 1b).The branching characteristics revealed that BcABF1 and BnABF1 are on the same evolutionary branch, indicating a close genetic relationship between B. campestris and B. napus.

Expression Analysis of BcABF1
To obtain the tissue-specific expression of BcABF1 in Pakchoi, qRT-PCR analysis was completed on the root, stem, leaf, stalk, petiole, and flower.Our findings revealed that the expression level of BcABF1 was the highest expression in the leaves, with the second highest expression observed in the flowers (Figure 2a).To advance our understanding of the BcABF1 expression pa ern, we engineered a GUS reporter system under the control of the BcABF1 promoter (pBcABF1:GUS) and transformed it into Arabidopsis thaliana (Col-0), resulting in the creation of the pBcABF1:GUS/Col.GUS staining of this line revealed a notable staining intensity in the leaves, primarily concentrated in the vascular and stoma (Figure 2b).Previous research has documented diurnal rhythm variations in ABF [43].We conducted a qRT-PCR analysis to assess the diurnal variations in ABF1 expression over a 24 h period in 14 d Pakchoi seedlings (Figure 2c).This analysis revealed that the expression of BcABF1 was significantly higher during daylight hours compared to nigh ime.Notably, the expression peaked at Zeitgeber Time (ZT) 8. Following this peak, there was a gradual decrease in expression.Subsequently, to explore ABF1's responsiveness to ABA, we applied 50 µM ABA to 14-day Pakchoi seedlings at ZT0.Our findings revealed that, relative to untreated Pakchoi, BcABF1's expression levels surged within the initial 8 h window post-treatment, diminished noticeably from 12 to 24 h, and eventually stabilized at an elevated level by the 24 h mark.Specifically, for post-ABA treatment, the most substantial shift in BcABF1 expression occurred at ZT8, where it was threefold higher than that in untreated plants (Figure 2c).These results indicate that BcABF1 is primarily expressed in leaves and is ABAinducible, displaying a strict diurnal rhythm change.

Overexpression of BcABF1 in Arabidopsis Reduces Leaf Water Loss
To study the function of BcABF1, we engineered the 35S:BcABF1 expression vector and successfully transformed it into Arabidopsis, leading to the creation of stable 35S:BcABF1 lines (#1, #2) in Col-0 (Figures 3a and S1a).Following qRT-PCR analysis, the expression level of BcABF1 was found to be higher in line #2.Hence, line #2 was selected.Intriguingly, we noticed an increase in the overall leaf size of the 35S:BcABF1 (Figure 3a).To understand the factors driving this morphological alteration in 35S:BcABF1, we measured the dimensions of the largest leaf in 28-day 35S:BcABF1 and WT.While the WT had a leaf length of 51.40 mm and a width of 21.75 mm, the 35S:BcABF1 exhibited significantly larger leaves, with lengths and widths measuring 63.85 mm and 24.60 mm, respectively (Figure 3b,c).This clear disparity in leaf size, with 35S:BcABF1 leaves being notably longer and wider than those of the WT.To verify this finding, we compared the fresh and dry weights of the 35S:BcABF1 and WT.The analysis revealed that 35S:BcABF1 displayed an increase in both fresh and dry weights in contrast to WT (Figure S1b,c).These findings indicate that the heterologous expression of BcABF1 in Arabidopsis promotes the growth of plants.Studies have demonstrated the critical role of auxins in the growth and development of plants [44].Among them, Indole-3-acetic acid (IAA) stands out not only as the principal auxin but also as one of the auxins with the highest physiological activity [45].Further, utilizing the ELISA technique, we quantified the IAA content in 28-day 35S:BcABF1 and WT.The IAA concentration in WT was found to be 617 ng/g, whereas a marked increase to 648 ng/g was observed in 35S:BcABF1 (Figure S1d).These findings imply a possible connection between BcABF1 and auxin levels.These results indicate that BcABF1 is primarily expressed in leaves and is ABA-inducible, displaying a strict diurnal rhythm change.

Overexpression of BcABF1 in Arabidopsis Reduces Leaf Water Loss
To study the function of BcABF1, we engineered the 35S:BcABF1 expression vector and successfully transformed it into Arabidopsis, leading to the creation of stable 35S:BcABF1 lines (#1, #2) in Col-0 (Figures 3a and S1a).Following qRT-PCR analysis, the expression level of BcABF1 was found to be higher in line #2.Hence, line #2 was selected.Intriguingly, we noticed an increase in the overall leaf size of the 35S:BcABF1 (Figure 3a).To understand the factors driving this morphological alteration in 35S:BcABF1, we measured the dimensions of the largest leaf in 28-day 35S:BcABF1 and WT.While the WT had a leaf length of 51.40 mm and a width of 21.75 mm, the 35S:BcABF1 exhibited significantly larger leaves, with lengths and widths measuring 63.85 mm and 24.60 mm, respectively (Figure 3b,c).This clear disparity in leaf size, with 35S:BcABF1 leaves being notably longer and wider than those of the WT.To verify this finding, we compared the fresh and dry weights of the 35S:BcABF1 and WT.The analysis revealed that 35S:BcABF1 displayed an increase in both fresh and dry weights in contrast to WT (Figure S1b,c).These findings indicate that the heterologous expression of BcABF1 in Arabidopsis promotes the growth of plants.Studies have demonstrated the critical role of auxins in the growth and development of plants [44].Among them, Indole-3-acetic acid (IAA) stands out not only as the principal auxin but also as one of the auxins with the highest physiological activity [45].Further, utilizing the ELISA technique, we quantified the IAA content in 28-day 35S:BcABF1 and WT.The IAA concentration in WT was found to be 617 ng/g, whereas a marked increase to 648 ng/g was observed in 35S:BcABF1 (Figure S1d).These findings imply a possible connection between BcABF1 and auxin levels.Studies have demonstrated that ABRE/ABF signaling can upregulate the expression of ADF5, which contributes to the remodeling of the actin cytoskeleton, thereby promoting the closure of stoma and subsequently enhancing the drought tolerance of plants [23].In an effort to investigate the effects of BcABF1 expression in stomata on the drought resistance capabilities of plants, we conducted an assessment of the leaf water loss rate in 21-day Arabidopsis.This involved specifically analyzing the aerial parts of the plants.Notably, when compared to the WT, the 35S:BcABF1 (#2) variant exhibited a substantially lower rate of leaf water loss at the 60 and 80 min marks during the dehydration process, highlighting its enhanced drought resilience.As expected, the detached leaves of 35S:BcABF1 exhibited significantly reduced water loss compared to WT (Figure 3d).To delve deeper into the impact of BcABF1 on enhancing plant drought resistance, specifically via the reduction in leaf water loss, we concentrated our efforts on assessing the expression of ABA-responsive genes that are known to be associated with drought resistance.These genes include RD29A, RD29B, RAB18, and KIN2 [46].Our investigation involved comparing these genes in both WT and 35S:BcABF1.We observed that the expression levels of RD29A, RD29B, RAB18, and KIN2 in the 35S:BcABF1 were significantly elevated in comparison to the WT (Figure 3e-h).These results further indicate that BcABF1 can improve plant drought resistance by reducing leaf water loss.

BcABF1 Directly Binds to the BcPYL4 Promoter
ABFs, key transcription factors in the ABA signaling pathway, are located downstream and primarily regulate the expression of ABA-responsive genes.Interestingly, in 35S:BcABF1, the expression of BcPYL4 was increased (Figure 4a).PYL4, known as a crucial member of the PYR/PYL receptor family, is located upstream of ABF and plays a vital role in maintaining the normal operation of the ABA signaling pathway [47,48].We assumed that ABF1 might be involved in feedback regulating the expression of PYL4, thereby modulating the ABA signaling pathway.To test this hypothesis, we analyzed the BcPYL4 2000 bp promoter and found the presence of an ABRE element (−916) (Figure S2a).Next, we used the Yeast One-Hybrid Assay (Y1H) to check whether BcABF1 binds to the BcPYL4 promoter region.The assay showed that BcABF1 can directly bind to the BcPYL4 promoter (Figure 4b).Furthermore, we conducted an analysis on the promoter cis elements of additional PYR/PYL/RCAR receptor family members.Our findings revealed the presence of ABRE binding sites on the promoters of BcPYR1, BcPYL2, BcPYL3, BcPYL5, BcPYL7, BcPYL8, and BcPYL11 (Figure S2a).However, using Y1H analysis to further study the interaction between BcABF1 and these promoters, we discovered that BcABF1 can only bind to the promoter of BcPYL2 (Figure S2b).
Subsequently, to delve deeper into BcABF1 ′ s regulatory role on BcPYL4, a dualluciferase assay was executed.Notably, the LUC/REN ratio in plants co-transformed with 35S:BcABF1-GFP and pBcPYL4-3-0800 demonstrated a significant increase compared to the control (Figure 4c,d).This suggests that BcABF1 actively binds to the ABRE motifs in the BcPYL4 promoter, thereby enhancing its expression (Figure 4c,d).Subsequently, our focus shifted to assessing whether BcABF1 could activate BcPYL4 within a living organism.For this purpose, a GUS (β-glucuronidase) reporter gene, driven by the BcPYL4 promoter (pBcPYL4:GUS), was constructed and introduced into Col-0 and 35S:BcABF1 Arabidopsis.In line with our predictions, the pBcPYL4:GUS signal was observable in the stoma of pBcPYL4:GUS/Col.More strikingly, GUS staining was markedly more intense in the 35S:BcABF1/pBcPYL4:GUS line compared to the control pBcPYL4:GUS/Col (Figure 4e).Furthermore, there was a significant increase in GUS activity in the 35S:BcABF1/pBcPYL4:GUS (Figure 4f).These findings lead us to conclude that BcABF1 is capable of directly binding to the BcPYL4 promoter, thereby activating its transcription. of pBcPYL4:GUS/Col.More strikingly, GUS staining was markedly more intense in 35S:BcABF1/pBcPYL4:GUS line compared to the control pBcPYL4:GUS/Col (Figure Furthermore, there was a significant increase in GUS activity in 35S:BcABF1/pBcPYL4:GUS (Figure 4f).These findings lead us to conclude that BcAB capable of directly binding to the BcPYL4 promoter, thereby activating its transcripti

Overexpression of BcPYL4 Amplifies the ABA Signaling Pathway
To further investigate the role of BcPYL4 in the ABA signaling pathway, a stabl abidopsis line expressing 35S:BcPYL4 was established (#7, #8, and #12) (Figure 5a,b) also conducted leaf water loss experiments on three lines of 35S:BcPYL4 (Figure 5c) results indicated that there was a reduction in the rate of leaf water loss in these three compared to WT, especially at 60 min and 100 min.This is similar to the dehydr phenotype of our BcABF1 overexpressing Arabidopsis.To advance our exploratio BcPYL4′s role in enhancing drought resistance, we conducted an analysis focusing o expression of drought response genes RD29A and RAB18 (Figure 5d,e).In the 35S:BcP strains, there was a significant increase in the expression levels of RD29A and RA reaching ten times that of the WT.The gene expression pa ern observed in 35S:BcP closely parallels that seen in 35S:BcABF1, indicating a uniform enhancement of dro resistance mechanisms facilitated by BcPYL4.

Overexpression of BcPYL4 Amplifies the ABA Signaling Pathway
To further investigate the role of BcPYL4 in the ABA signaling pathway, a stable Arabidopsis line expressing 35S:BcPYL4 was established (#7, #8, and #12) (Figure 5a,b).We also conducted leaf water loss experiments on three lines of 35S:BcPYL4 (Figure 5c).The results indicated that there was a reduction in the rate of leaf water loss in these three lines compared to WT, especially at 60 min and 100 min.This is similar to the dehydration phenotype of our BcABF1 overexpressing Arabidopsis.To advance our exploration of BcPYL4 ′ s role in enhancing drought resistance, we conducted an analysis focusing on the expression of drought response genes RD29A and RAB18 (Figure 5d,e).In the 35S:BcPYL4 strains, there was a significant increase in the expression levels of RD29A and RAB18, reaching ten times that of the WT.The gene expression pattern observed in 35S:BcPYL4 closely parallels that seen in 35S:BcABF1, indicating a uniform enhancement of drought resistance mechanisms facilitated by BcPYL4.
Moreover, the ABA signaling pathway not only regulates plant drought resistance but also plays a role in leaf aging, flowering, and seed germination processes [49].Subsequent analysis of the expression changes in leaf aging genes (NYC1 and NYE1), flowering-related gene (SOC1), and seed germination-related gene (CYS5) in 35S:BcPYL4 showed an upregulation of these response genes (Figure 5f-i).These indicate that overexpression of BcPYL4 amplifies the ABA signaling pathway, not only enhancing plant drought resistance but also participating in the regulation of ABA-induced leaf aging, flowering, and seed germination processes.Moreover, the ABA signaling pathway not only regulates drought resistance but also plays a role in leaf aging, flowering, and seed germination processes [49].Subsequent analysis of the expression changes in leaf aging genes (NYC1 and NYE1), floweringrelated gene (SOC1), and seed germination-related gene (CYS5) in 35S:BcPYL4 showed an upregulation of these response genes (Figure 5f-i).These indicate that overexpression of BcPYL4 amplifies the ABA signaling pathway, not only enhancing plant drought resistance but also participating in the regulation of ABA-induced leaf aging, flowering, and seed germination processes.

Discussion
ABF1, a member of the bZIP family, exhibits a high degree of conservation across plant species and features the BRLZ domain [50].In our study, we explored ABF1 proteins across seven cruciferous plant species.Our findings revealed that ABF1 shares similar structural features, characterized by a BRLZ domain present in all ABF1 proteins.Notably,

Discussion
ABF1, a member of the bZIP family, exhibits a high degree of conservation across plant species and features the BRLZ domain [50].In our study, we explored ABF1 proteins across seven cruciferous plant species.Our findings revealed that ABF1 shares similar structural features, characterized by a BRLZ domain present in all ABF1 proteins.Notably, a close evolutionary relationship between B. campestris and B. napus was observed (Figure 1), highlighting evolutionary similarities in ABF1 functions.In Arabidopsis, the expression of all ABFs is induced by ABA, drought, and low-temperature conditions [51].Remarkably, BcABF1 is also ABA-induced and primarily expressed in stomata and vascular tissues (Figure 2).Furthermore, ABFs exhibit diverse functions in evolution; studies suggest that ABF2, ABF3, and ABF4 play pivotal roles in regulating chlorophyll degradation and leaf senescence processes [52].In this study, we identified the positive impact of BcABF1 on reducing leaf water loss and orchestrating the expression of ABA-responsive genes during this process (Figure 3).The mechanism by which BcABF1 reduces leaf water loss remains to be fully understood.It has been reported that ABFs play a positive role in the regulation of stomatal closure.The interaction between ABF1 and IDD14 is implicated in the modulation of stomatal closure, resulting in a further decrease in leaf surface water loss and, to some extent, enhancing drought resistance in Arabidopsis [22].Overexpression of ABF3 and ABF4 induces partial stomatal closure, leading to a reduction in water loss [24].ANAC096, interacting with ABF2 and ABF4, actively participates in the regulation of ABA-mediated stomatal closure and water loss, further enhancing plant drought resistance [27].Therefore, based on our data, we have reason to believe that the reduction in leaf water loss by BcABF1 is mediated via the regulation of stomatal closure.However, the specific regulatory mechanism still requires further exploration.
Additionally, our data indicate that the overexpression of BcABF1 in Arabidopsis leads to an enlarged phenotype (Figure 3a-c).These findings are intriguing because, in recent research, the overexpression of bZIP transcription factors often results in impaired plant growth.For instance, overexpression of Osbzip48 significantly reduces plant height, ascribed to the direct binding of Osbzip48 to Osko2 and the subsequent regulation of its expression, causing dwarfism in rice (Oryza sativa L.) [53].Similarly, overexpression of OsABF1 in rice also exhibits a semi-dwarf phenotype [54].Another study reports that transgenic Arabidopsis expressing TaABF3 shows a slight reduction in the size of rosette leaves during growth [50].It is worth noting that genetic functional diversity arising from species genomic differences can result in phenotypic variations [55].In our data, the overexpression of BcABF1 in Arabidopsis displays an enlarged phenotype (Figure 3a-c).In this regard, we explored potential causes.According to pertinent studies, auxin plays a crucial role in plant growth and development [44].Indole-3-acetic acid (IAA) serves not only as a major auxin but is also one of the most physiologically active auxins [45].Therefore, we measured the IAA content in 35S:BcABF1 (Figure S1d).Our results show a significant increase in IAA content in 35S:BcABF1, providing an explanation for the observed enlarged phenotype.Studies have indicated that ABFs interact with IDD14, regulating the ABA signaling pathway [22].Simultaneously, IDD14 directly activates TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1, PIN-FORMED1, and YUCCA5, thereby regulating auxin synthesis and transport [56].Moreover, ABA modulates the auxin signaling pathway by regulating auxin response factors ARF5, ARF6, and ARF10 [57].The wide-ranging interplay and crosstalk between ABA and IAA in the regulation of stress responses are well-established [58].Under stressful conditions, increased ABA levels suppress the expression of genes encoding proteins involved in auxin transport.Conversely, under non-stressful conditions, low ABA levels stimulate auxin transport, initiating the auxin signaling pathway [59].During normal growth, low ABA levels facilitate auxin transport, aiding in the accumulation of IAA in leaves and promoting leaf expansion in 35S:BcABF1.Hence, we have reason to believe that BcABF1 serves as a key junction connecting ABA and IAA hormonal signals, modulating critical nodes in plant growth and development.
The transcription factor ABF plays a pivotal regulatory role in the ABA signaling pathway, and extensive research has been carried out on ABF at both the transcriptional and translational levels [12,27,60,61].However, there is a limited body of research exploring how ABF contributes to feedback regulation in the ABA signal.Currently, molecular mechanisms of ABA signaling have been extensively investigated.The fundamental components of the ABA signaling pathway encompass ABA receptors, PP2Cs, and SnRK2s [12,62].In the context of feedback regulation in the ABA signal transduction pathway, ABF is recognized as a crucial regulatory factor.PP2C, acting as a negative regulator of ABA signaling, induces the expression of ABFs, leading to their dephosphorylation and inactivation, thus maintaining the equilibrium of the ABA signal [32].ABF4 directly activates FYVE1, and FYVE1 binds and degrades PYL, accomplishing negative feedback regulation of the ABA signal [36].Moreover, FYVE1 can be phosphorylated by SnRK2, and the phosphorylated FYVE1 inhibits the transcriptional activation activity of ABF4 [36].Our data provide novel insights into the feedback regulation of ABA.BcABF1, by directly binding to the BcPYL4 promoter, enhances BcPYL4 expression, thus increasing ABA receptor levels (Figure 4).Overexpressing BcPYL4 in Arabidopsis reduces leaf water loss and upregulates ABA-responsive gene expression (Figure 5).Consequently, we propose that BcABF1, via BcPYL4 activation, participates in the positive feedback loop of ABA signaling.While there is functional redundancy among the 14 PYLs [37], not all are involved in ABF-mediated ABA feedback regulation; in Pakchoi, only BcPYL4 and BcPYL2 play a role (Figures 4 and S2).Therefore, our data unveil an alternative regulatory pathway for BcABF1 in ABA signaling.In this context, we establish a regulatory mechanism in plants to swiftly adapt to environmental stress by amplifying the ABA signal.BcABF1 serves as a positive feedback regulator in the ABA signaling pathway, enhancing the ABA signal by increasing ABA receptor levels.Simultaneously, to prevent excessive response or ABA accumulation, ABF functions as a negative feedback regulator.It achieves this by inducing PP2C expression and degrading PYL, strictly maintaining ABA signal homeostasis.

Plant Materials and Growth Conditions
The Pakchoi used in this study were sourced from the Cabbage Systems Biology Laboratory at the College of Horticulture, Nanjing Agricultural University.Pakchoi, Arabidopsis thaliana of the 'Columbia-0' ecotype (Col-0), transgenic Arabidopsis, and N. benthamiana were all grown under conditions of 23 • C and a long-day photoperiod (16 h of light/8 h of dark).Col-0 and Pakchoi served as controls in our experiments, except where it was noted otherwise.

Arabidopsis Transformation
Using Pakchoi cDNA as a template, the coding sequences (CDS) of BcABF1 and BcPYL4 were amplified.The BcABF1 CDS was then cloned into the pMDC43 vector to obtain the 35S:BcABF1 expression vector.The BcPYL4 CDS was inserted into the pRI101 vector to obtain the 35S:PYL4 expression vector.Sequencing confirmed the correctness of both expression vectors.
The 35S:BcABF1 and 35S:BcPYL4 expression vectors were separately introduced into Agrobacterium (GV3101) and transformed into Col-0 using the floral dip method [63].T 3 generation homozygous overexpressing Arabidopsis lines were obtained via screening.The primers used for amplifying sequences are listed in Table S1.

β-Glucuronidase Staining and Expression
Using Pakchoi genomic DNA as a template, a 2000 bp fragment of the upstream 5 ′ region of the BcPYL4 gene was amplified.This promoter fragment was then cloned into the PBI121 expression vector to generate the BcPYL4:GUS expression vector (primers listed in Table S1).After sequencing confirmation, the vector was introduced into Agrobacterium (GV3101) and separately transformed into T 3 generation homozygous overexpressing 35S:BcABF1 lines and Col-0 using the floral dip method [63].Selection using kanamycin on the growth medium resulted in the acquisition of homozygous T 3 lines for pBcPYL4:GUS/Col and pBcPYL4:GUS/35S:BcABF1.As previously mentioned, the GUS enzyme activity was measured [64].

Yeast One-Hybrid Assay (Y1H)
To investigate the interaction between BcABF1 and the BcPYL4 promoter, the Match-maker™ Gold Yeast One-Hybrid System from Clontech was employed for a yeast onehybrid assay (Y1H).For this purpose, BcABF1 was integrated into the pGADT7 vector, while the 2 kb promoter region of BcPYL4 was cloned into the pAbAi vector.After screening for suitable AbA concentrations, the interaction between pAbAi-BcPYL4 and BcABF1 was validated using the lithium acetate method (Clontech, Mountain View, CA, USA, Cat.630439).
Genes homologous to the Arabidopsis PYR/PYLs family were extracted from the Pakchoi genome and assessed for their interaction with BcABF1 at the BcPYLs promoters using the Yeast One-Hybrid (Y1H) assay.The methodology employed for this verification was consistent with the previously described approach.

Dual-Luciferase Reporter Assay
The BcABF1 gene sequence was inserted into the pRI101 vector, with 35S:BcABF1-GFP and 35S:GFP serving as effector vectors.The BcPYL4 promoter was inserted into the pGreenII-0800-LUC vector as a reporter vector (primers listed in Table S1).Following a 2:1 ratio of reporter vector to effector vector, the mixture was infiltrated into tobacco leaves.After 72 h, D-luciferin was injected into the tobacco leaves, followed by a 5 min dark incubation.Fluorescence intensity was then measured using a live plant imaging system (LUC).The LUC/REN ratio reflects binding activity.

Determination of IAA Content
An amount of 0.1 g of fresh Arabidopsis leaves from 28-day WT and 35S:BcABF1 were ground thoroughly with liquid nitrogen, and 900 µL of PBS buffer was added and mixed thoroughly.It was centrifuged at 4 • C and 8000 rpm for 30 min, and the supernatant was used for further analysis.The IAA content is measured using the Auxin ELISA Kit (BYabscience (Nanjing) Co., Ltd., Nanjing, China) according to the instructions.In the enzyme-linked immunosorbent assay (ELISA) approach, we blend a specific quantity of solid-phase antibodies with biotin-tagged IAA (indole-3-acetic acid), Auxin and non-labeled antigens (either calibration standards or test samples), to initiate a competitive inhibition reaction.Once the reaction achieves equilibrium, a complex of solid-phase antibodies linked to biotinylated IAA, Auxin is formed.Subsequently, enzyme-tagged avidin is introduced, resulting in the formation of a complex involving solid-phase antibody-biotinylated IAA, Auxin bound to enzyme-labeled avidin.Following the color development of the substrate, the absorbance (OD value) is measured at a 450 nm wavelength using an ELISA reader, providing valuable insights into the assay's outcomes.

Leaf Water Loss
To accurately assess the rate of leaf water loss, we chose aerial sections from 35S:BcABF1, 35S:BcPYL4, and WT, ensuring they were of comparable size and at a uniform developmental stage.Each sample was composed of the aerial parts from 10 individual plants.These samples were cultivated under conditions mirroring those of plant growth.Subsequently, each sample was weighed at specific intervals (0, 20, 40, 60, 80, 100, and 120 min).Leaf water loss was quantified by the reduction in fresh weight, and the leaf water loss rate was calculated using the formula: (fresh weight − dry weight)/fresh weight, expressed as a percentage.This method provides a precise measure of water loss in leaves under varying conditions.

Statistical Analysis
All experiments were conducted independently at least three times, with statistical analysis of the data performed using Student's t-test.p < 0.05 or p < 0.01.

Figure 1 .
Figure 1.Phylogenetic analysis of ABF1.(a) Amino acid sequence alignment of ABF1 in seven cruciferous plant species.Identical and similar amino acids are indicated with black and pink shading, respectively, while highly variable amino acids are represented with blue shading.The part marked by the green rectangle is the BRLZ domain.(b) Phylogenetic tree.Phylogenetic tree was constructed using MEGA X (neighbor-joining algorithm, bootstrap replications = 1000).The numbers beside the branches represent bootstrap values from 1000 replicates.Relative changes along the branches are indicated by the scale bar.Bc: Brassica campestris.Bna: Brassica napus.Bju: Brassica juncea.Bni: Brassica nigra.Bol: Brassica oleracea.Bca: Brassica carinata.At: Arabidopsis thaliana.

Figure 1 .
Figure 1.Phylogenetic analysis of ABF1.(a) Amino acid sequence alignment of ABF1 in seven cruciferous plant species.Identical and similar amino acids are indicated with black and pink shading, respectively, while highly variable amino acids are represented with blue shading.The part marked by the green rectangle is the BRLZ domain.(b) Phylogenetic tree.Phylogenetic tree was constructed using MEGA X (neighbor-joining algorithm, bootstrap replications = 1000).The numbers beside the branches represent bootstrap values from 1000 replicates.Relative changes along the branches are indicated by the scale bar.Bc: Brassica campestris.Bna: Brassica napus.Bju: Brassica juncea.Bni: Brassica nigra.Bol: Brassica oleracea.Bca: Brassica carinata.At: Arabidopsis thaliana.

Figure 2 .
Figure 2. BcABF1 expression analysis.(a) Expression levels of BcABF1 in different tissues of Pakchoi.(b) GUS staining of pBcABF1:GUS/Col.(c) Pakchoi seedlings were treated with 50 µM ABA under long-day conditions (LD), with spraying twice a week for two consecutive weeks.Samples were collected every 4 h starting from ZT0. qPT-PCR analysis was conducted to measure the expression levels of BcABF1.The black areas represent dark conditions, while the white areas indicate light

Figure 2 .
Figure 2. BcABF1 expression analysis.(a) Expression levels of BcABF1 in different tissues of Pakchoi.(b) GUS staining of pBcABF1:GUS/Col.(c) Pakchoi seedlings were treated with 50 µM ABA under long-day conditions (LD), with spraying twice a week for two consecutive weeks.Samples were collected every 4 h starting from ZT0. qPT-PCR analysis was conducted to measure the expression levels of BcABF1.The black areas represent dark conditions, while the white areas indicate light conditions.The data represent the average of three biological replicates (** p < 0.01, * p < 0.05, Student's t-test).

Figure 3 .
Figure 3. Phenotypic indicators analysis of BcABF1 overexpressing Arabidopsis.(a) The phenotype of Arabidopsis overexpressing 35S:BcABF1.(b) Leaf length in 35S:BcABF1.The largest leaf from each individual 28-day 35S:BcABF1 and WT was selected and identified, and then its length was accurately measured.(c) Leaf width in 35S:BcABF1.The largest leaf from each individual 28-day 35S:BcABF1 and WT was selected and identified and then its width was accurately measured.(d) Rate of water loss in leaves of 35S:BcABF1.Leaves from 21-day-old 35S:BcABF1 were selected, and measurements were taken every 20 min.(e-h) Relative expression levels of ABA signaling response genes in 35S:BcABF1.The data represent the average of three biological replicates (* p < 0.05, Student's t-test).
To elucidate the evolutionary relationship of ABF1, we selected seven cruciferous plant species (B.napus, B. campestris, B. carinata, B. nigra, B. juncea, B. oleracea, and Arabidopsis thaliana) for analysis.We used DNAMAN for amino acid sequence alignment and the SMART online analysis tool for the prediction of functional structures.Specifically, the amino acid sequence of ABF1 in B. campestris was found to be 82.68%similar to B. oleracea, 96.73% to B. napus, 96.46% to both B. nigra and B. juncea, 89.3% to B. carinata, and 73.53% to Arabidopsis thaliana (Figure amino acid sequence of ABF1 in B. campestris was found to be 82.68%similar to B. oleracea, 96.73% to B. napus, 96.46% to both B. nigra and B. juncea, 89.3% to B. carinata, and 73.53% to Arabidopsis thaliana (Figure