Natural variation in BnaA9.NF-YA7 contributes to drought tolerance in Brassica napus L

Wang, Jia; Mao, Lin; Li, Yangyang; Lu, Kun; Qu, Cunmin; Tang, Zhanglin; Li, Jiana; Liu, Liezhao

doi:10.1038/s41467-024-46271-2

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Published: 07 March 2024

Natural variation in BnaA9.NF-YA7 contributes to drought tolerance in Brassica napus L

Nature Communications volume 15, Article number: 2082 (2024) Cite this article

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Abstract

Rapeseed (Brassica napus) is one of the important oil crops worldwide. Its production is often threatened by drought stress. Here, we identify a transcription factor (BnaA9.NF-YA7) that negatively regulates drought tolerance through genome-wide association study in B. napus. The presence of two SNPs within a CCAAT cis element leads to downregulation of BnaA9.NF-YA7 expression. In addition, the M63I (G-to-C) substitution in the transactivation domain can activate low level expression of BnaA4.DOR, which is an inhibitory factor of ABA-induced stomatal closure. Furthermore, we determine that Bna.ABF3/4s directly regulate the expression of BnaA9.NF-YA7, and BnaA9.NF-YA7 indirectly suppresses the expression of Bna.ABF3/4s by regulation of Bna.ASHH4s. Our findings uncover that BnaA9.NF-YA7 serves as a supplementary role for ABA signal balance under drought stress conditions, and provide a potential molecular target to breed drought-tolerant B. napus cultivars.

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Introduction

Brassica napus L. (B. napus; AACC, 2n = 38), an amphidiploid species formed by the natural hybridization of Brassica rapa (AA, 2n = 20) and Brassica oleracea (CC, 2n = 18)¹, is an excellent source of edible vegetable oil and protein and constitutes the second-largest vegetable oil source in the world^2,3,4. However, drought stress has a major limiting impact on its production and yield stability⁵, and drought accounts for at least a 30% yield loss in B. napus every year⁶. Therefore, exploring the mechanisms by which B. napus responds to drought stresses and how the resistance of B. napus to drought stress may be improved has great significance. To date, some drought-responsive genes, including LEA3 and VOC (which alter drought tolerance and lipid accumulation²), BnaYUC6a (which is associated with high auxin production and drought tolerance⁷), BnaA6.RGA (which enhances drought tolerance⁸), and BnaA01.CIPK6 (which confers drought tolerance⁹), have been cloned and functionally characterized in B. napus. However, the molecular regulation of drought tolerance in B. napus is largely unknown.

Nuclear factor-Y (NF-Y), also known as heme activator protein (HAP) or CCAAT-binding factor (CBF), is a sequence-specific transcription factor with nonspecific DNA-binding nucleosome properties that is ubiquitous in eukaryotes, and is composed of NF-YA, NF-YB, and NF-YC subunits^10,11. The three subunits can form a heterotrimeric complex and bind to the downstream gene CCAAT-box, one of the sequence elements most frequently found in eukaryotic promoters, and regulate its expression¹¹. In recent years, many studies have found that NF-Y transcription factors in plants play an important role in plants’ ability to cope under various abiotic stresses, including drought stress^12,13,14,15. From a functional viewpoint, NF-Y is considered a facilitator of TF binding and an architectural promoter organizer¹¹. Furthermore, NF-Y complex binding to DNA is also important for the establishment of histone posttranslational modifications (PTMs) through the recruitment of relevant enzymes¹⁶.

Here, we report that an NF-YA subunit, BnaA9.NF-YA7, is involved in B. napus drought tolerance at the seedling stage. Two SNPs in the promoter and a nonsynonymous substitution (M63I) in BnaA9.NF-YA7 confer B. napus drought tolerance by regulating the ABA signal transduction pathway via feedback inhibition of the expression of BnaABF3/4s-related genes.

Results

Natural variation in BnaA9.NF-YA7 is associated with drought tolerance

Further mining of previously published data¹⁷ showed that BnaA9.NF-YA7 is associated with the germination percentage (GP) and germination index (GI) under drought stress and that BnaA9.NF-YA7 is upregulated by drought stress under repeated drought-rewatering cycles (Supplementary Fig. 1). To investigate functional allelic variations in the BnaA9.NF-YA7 locus, 48 variation sites covering the BnaA9.NF-YA7 promoter (2 kb) and gene were identified based on previously published resequencing data¹⁸ (Supplementary Data 1). Candidate gene association analysis was performed for GP and GI using 181 inbred lines from the resequencing population and five SNPs were identified above the suggestive significance threshold of association (P < 0.01; Supplementary Fig. 2a, b). Moreover, GWAS performed in this natural-variation population for water holding capacity (WHC) under drought stress using a mixed linear model approach identified ten associated loci meeting the threshold for suggestive evidence of association (P < 1.7 × 10⁻⁷), of which the most significant signal (S9_19159584) was located within the third exon of BnaA9.NF-YA7 (Fig. 1a and Supplementary Fig. 2c).

Interestingly, S9_19159584 (G-to-C), in which the methionine at the 63rd position was changed to isoleucine (M63I) in BnaA9.NF-YA7, was in strong linkage disequilibrium (LD) with the SNPs within the promoter region (Fig. 1b). The 608 rapeseed genotypes were classified into five haplotype groups (Supplementary Fig. 2d), based on the SNPs within BnaA9.NF-YA7 (MAF ≥0.03), and physiologic index analysis showed significant differences among different haplotypes, especially Hap3 and Hap4 (Fig. 1c, d and Supplementary Fig. 2e–h). Moreover, the accessions with the C allele (Hap1 and Hap4, referred to as BnaA9.NF-YA7^C) had a higher WHC than the accessions with the G allele (referred to as BnaA9.NF-YA7^G) after drought treatment (Fig. 1e and Supplementary Fig. 2i). However, no mutation sites significantly associated with drought tolerance were found in another homolog BnaC5.NF-YA7 in B. napus (Supplementary Fig. 2c). Together, these results suggest the genetic variation in BnaA9.NF-YA7 could be involved in the drought tolerance of B. napus.

Next, we performed multiple sequence alignments and phylogenetic tree analysis of the BnaA9.NF-YA7 protein sequence. Surprisingly, the genotypes of Bra.NF-YA7, Bol.NF-YA7 and BnaC5.NF-YA7 at position 63 were consistent with BnaA9.NF-YA7^C (Supplementary Fig. 3a), and BnaA9.NF-YA7^C was evolutionarily closer to Bra.NF-YA7 (Supplementary Fig. 3b), indicating that only 10% (52/608) of the C alleles in B. napus were from ancestral species. Nevertheless, the M63I substitution did not change the tertiary structure of the protein (Supplementary Fig. 3c). Several BnaA9.NF-YA7 fragments were designed based on the domain structure of BnaA9.NF-YA7 (Supplementary Fig. 3d). The Y2H (self-activation) assay and dual-luciferase reporter assay showed that BnaA9.NF-YA7 had different transcriptional activation abilities in Hap3 and Hap4 (Supplementary Fig. 3e–h), and the M63I substitution occurred in a Pro-rich transcriptional activation domain (Supplementary Fig. 3a). Unlike the Hap1 varieties, the Hap4 members were clustered in a small subgroup of the semi-winter ecotype (Supplementary Fig. 4a). The highest F_ST levels appeared at the site in BnaA9.NF-YA7 and its flank but not in BnaC5.NF-YA7 (Fig. 1f and Supplementary Fig. 4b), suggesting that BnaA9.NF-YA7 possibly underwent strong selection during the domestication stage.

BnaA9.NF-YA7 was greatly induced by drought and abscisic acid

Under nonstress conditions, higher levels of BnaA9.NF-YA7 transcripts were found in embryos, leaves, and seeds compared with other tissues, similar to the result for BnaC5.NF-YA7 (Supplementary Fig. 5a). BnaA9.NF-YA7 expression was significantly induced by drought stress, and the expression level of Hap3 was significantly higher than that of Hap4 (Supplementary Fig. 5b). The same phenomenon occurred under osmotic stress simulated by 0.2 M d-mannitol and 15% PEG, with a negative correlation between survival rate and expression level, which was almost the same as the result under drought treatment (Fig. 1g, h and Supplementary Fig. 5c, d). Unexpectedly, the expression level of Hap1 was also higher than that of Hap4 (Fig. 1g). Moreover, the expression of BnaC5.NF-YA7 was also induced by drought, but the level of BnaC5.NF-YA7 was not significantly different among the five haplotypes (Supplementary Fig. 5e).

Compared to the unaltered transcripts in water-treated leaves (Supplementary Fig. 6a), the expression of BnaA9.NF-YA7 was upregulated by H₂O₂, ABA, and SNP (sodium nitroprusside, nitric oxide producer) treatment, and there were also differences between Hap3 and Hap4 (Supplementary Fig. 6b–d). Likewise, the expression of BnaA9.NF-YA7 was significantly induced after dehydration treatment (Supplementary Fig. 6e). Upregulation of BnaA9.NF-YA7 expression by ABA treatment and induction of BnaA9.NF-YA7 expression was suppressed by pretreatment with FLU (fluridone, an inhibitor of ABA biosynthesis) in detached leaves, suggesting that ABA was an essential signal for dehydration-induced transcription of BnaA9.NF-YA7 (Supplementary Fig. 6f). These findings indicated that BnaA9.NF-YA7 is responsive to drought stress and ABA signaling.

BnaA9.NF-YA7 negatively regulates B. napus drought tolerance

To confirm the function of BnaA9.NF-YA7, we generated transgenic B. napus plants (Supplementary Fig. 7). The results of drought tolerance evaluation showed that most of the knockout and RNAi seedlings survived after a 5-day recovery because of increased leaf water holding capacity and decreased water loss rate and transpiration rate (Fig. 2a–e and Supplementary Fig. 8a–e). A similar result was also observed for germination under d-mannitol treatment; the knockout seedlings exhibited increased root length and hypocotyl length, but the BnaA9.NF-YA7-OE lines had shorter roots and hypocotyls than the WT (Supplementary Fig. 8f–h). These results demonstrate that BnaA9.NF-YA7 functions as a negative regulator of B. napus drought tolerance. Furthermore, we noted that there was no significant difference in drought tolerance between the BnaA9.NF-YA7^C and BnaA9.NF-YA7^G-overexpressing transgenic lines (Fig. 2f), but the drought tolerance of Hap3 could be slightly enhanced by the site-directed mutation M63I (Supplementary Fig. 8i, j).

In comparison with wild-type plants, the aaCC (BnaA9.NF-YA7 knockout) and aacc (BnaA9.NF-YA7/BnaC5.NF-YA7 double knockout) lines had smaller stomata and lower stomatal density (Fig. 2g–k), exhibiting lower stomatal conductance and transpiration rate under nonstress conditions (Fig. 2l, m). Conversely, overexpressing BnaA9.NF-YA7 led to increased stomatal conductance and transpiration rate compared with those of the WT, but significant differences were not found among these transgenic plants after ABA treatment (Fig. 2l, m). Additional confirmation was obtained by using infrared thermal imaging to visualize the surface temperature of detached leaves, in which the leaf surface temperature of the overexpression lines was lower due to more water loss than that of the WT and knockout lines before dehydration treatment (Supplementary Fig. 8a and Supplementary Fig. 9a, b), while that of the knockout line was slightly higher than that of the WT after 2 h of dehydration (Supplementary Fig. 9c). ABA treatment significantly inhibited root and hypocotyl growth in all genotypes (Fig. 2n), and the inhibition degree significantly increased in BnaA9.NF-YA7-OE plants compared to the WT and BnaA9.NF-YA7-KO lines (Fig. 2o, p), indicating that overexpression of BnaA9.NF-YA7 increased ABA sensitivity. However, the percentage of closed stomata was significantly lower in BnaA9.NF-YA7-OE (19.68%) compared to WT (52.50%) or BnaA9.NF-YA7-KO (62.87%) leaves after drought treatment (Supplementary Fig. 9d, e), similar to the result for ABA treatment (Fig. 2q). These results indicated that BnaA9.NF-YA7 negatively regulates drought tolerance by increasing stomatal conductance and the transpiration rate.

CCAAT-box variation affects the expression level of BnaA9.NF-YA7

GUS activity was clearly detected in all tissues and organs except for the root tip with different degrees of expression; in particular, GUS staining was more vasculature-specific in seedlings (Supplementary Fig. 10). In 2-week-old plants, GUS activity and GUS expression levels in leaves were significantly different between Hap3 and Hap4 under abiotic stress (Fig. 3a–c). To accurately identify the specific mutation sites that led to the differences in Hap3 and Hap4 expression levels, we conducted promoter truncation analysis and found that the ABRE, CCAAT-box, and motif_sequence were the core cis-acting elements of the BnaA9.NF-YA7 promoter (Fig. 3d), but the presence of two SNPs led to the CCAAT-box deletion in only Hap4, not in the other haplotypes (Fig. 3e and Supplementary Fig. 11). These findings provide strong evidence that CCAAT-box variation is directly involved in the difference in BnaA9.NF-YA7 transcript levels between Hap4 and other haplotypes (including Hap1).

To confirm this hypothesis, expression analysis and clustering of all NF-Y subunits (41 putative BnaNF-YA, 43 putative BnaNF-YB, and 18 putative BnaNF-YC subunits) were performed under repeated drought-rewatering treatment, and the NF-YA/YB/YC subunits within the B2 subgroup to which BnaA9.NF-YA7 belongs were selected for interaction analysis (Supplementary Fig. 12a). Y2H and BiFC assays detected interactions between BnaA9.NF-YB2 and BnaA6.NF-YC9, BnaA9.NF-YB2, and BnaC5.NF-YC9 (Supplementary Fig. 12b, c), and the BnaA9.NF-YB2/BnaA6.NF-YC9 dimer formed a trimer with BnaA6.NF-YA5 and BnaC5.NF-YA9 by yeast three-hybrid (Y3H) analysis (Supplementary Fig. 12d). Next, transient dual-luciferase assays were performed in Arabidopsis protoplasts. When co-transforming with an empty effector construct, the LUC activity of the Hap3 reporter was not different from that of the Hap4 reporter (Fig. 3f, g). Co-expression of BnaA9.NF-YB2/BnaA6.NF-YC9 with BnaA6.NF-YA5 induced LUC activity in both the Hap3 and Hap4 reporters, similar to BnaA9.NF-YB2/BnaC5.NF-YC9 with BnaC5.NF-YA9, whereas the Hap4 reporter exhibited lower LUC activity than the Hap3 reporter (P < 0.001, Fig. 3f, g), which explained the difference in expression levels between Hap3 and Hap4. In addition, we found that BnaA9.NF-YA7 can induce higher LUC activity in the Hap3 reporter than in Hap4, and BnaA9.NF-YA7^G induced higher LUC activity than BnaA9.NF-YA7^C, which further confirms that the M63I substitution is in the transcriptional activation region.

BnaABF3/4s directly regulate the activity of BnaA9.NF-YA7

To explore the upstream regulators that regulate BnaA9.NF-YA7 expression under drought stress, we screened for proteins that interact with D5 (a fragment of the promoter of BnaA9.NF-YA7, Fig. 3d) using a Y1H assay with a cDNA library prepared from drought-treated B. napus leaves (Supplementary Table 1). Among the potential regulatory genes, BnaABF3/4s were particularly interesting because of the upregulated expression of BnaA9.NF-YA7 induced by ABA. Both the Y1H assay and electrophoretic mobility shift assay (EMSA) showed that BnaC7.ABF3 and BnaA3.ABF4 bound the ABRE sites in the BnaA9.NF-YA7 promoter in vitro (Fig. 4a–d). Furthermore, ChIP‒qPCR identified enrichment in the fragment spanning the ABRE sites in the BnaA9.NF-YA7 promoter (Fig. 4e–g). We thus believed it was possible that BnaC7.ABF3 and BnaA3.ABF4 regulated the expression of BnaA9.NF-YA7 under ABA induction.

**Fig. 4: *BnaABF3/4* s directly upregulates the transcription of *BnaA9.NF-YA7*.**

A previous study suggested that AtABF3 and AtABF4 interact in Arabidopsis¹⁹. We tested for an interaction between the BnaC7.ABF3 and BnaA3.ABF4 proteins before further determining how this circuit was regulated, and both the Y2H assay and BiFC assay showed that there was a physical interaction between BnaC7.ABF3 and BnaA3.ABF4 (Supplementary Fig. 13a, b). Transient expression assays showed that overexpressing BnaC7.ABF3 or BnaA3.ABF4 alone induced the LUC activity of both the Hap3 and Hap4 reporters, and co-expression of BnaC7.ABF3 with BnaA3.ABF4 further induced the activity of the two reporters, whereas there was no significant difference in LUC activity between the Hap3 and Hap4 reporters (Fig. 4h, i).

To further determine the regulatory relationship of BnaA9.NF-YA7 with BnaC7.ABF3, we examined their expression in the leaves of BnaC7.ABF3 transgenic plants. The expression level of BnaA9.NF-YA7 was significantly increased in the BnaC7.ABF3-OE lines, but the transcript was repressed in the BnaC7.ABF3-RNAi lines even after ABA treatment, whereas the expression of BnaC7.ABF3 was unaltered in the BnaA9.NF-YA7-RNAi line (Supplementary Fig. 13c). Together, these data clearly indicate that BnaA9.NF-YA7 functions downstream of BnaABF3/4s.

Identification of stress-related genes controlled by BnaA9.NF-YA7

To understand the different molecular mechanisms regulated by BnaA9.NF-YA7 under drought stress, we compared the transcriptomes of leaves from drought-stressed 4-week-old Hap3 and Hap4 plants using RNA-Seq. The DEGs were stratified into hormone-mediated signaling pathway, regulation of jasmonic acid-mediated signaling pathway, and response to abiotic stimulus (Supplementary Fig. 14a and Supplementary Data 2), indicating that the difference between Hap3 and Hap4 is involved in a diverse range of biological processes and molecular functions. Moreover, we conducted RNA-Seq analysis using WT, BnaA9.NF-YA7-OE, and BnaA9.NF-YA7-KO (aaCC, aacc, and AAcc homozygous mutant plants) seedlings after drought stress to explore the genome-wide transcriptional landscape dictated by BnaA9.NF-YA7. Through gene ontology (GO) analysis, these DEGs were found to be enriched in “response to water deprivation”, “response to abscisic acid”, “response to abiotic stimulus”, and “response to osmotic stress” (Supplementary Fig. 14b, c and Supplementary Data 3,4). Notably, Venn diagram analysis showed that aaCC and AAcc shared 4706 DEGs (Supplementary Fig. 14d), indicating that, to a great extent, BnaA9.NF-YA7 and BnaC5.NF-YA7 share similar downstream targets and plays largely redundant roles during these different biological processes. However, we also found that the DEGs specifically regulated by BnaA9.NF-YA7 or BnaC5.NF-YA7 were stratified into different sets of biological processes (Supplementary Fig. 14e), suggesting that BnaA9.NF-YA7 and BnaC5.NF-YA7 may have distinct roles due to functional differentiation.

To better identify direct BnaA9.NF-YA7-binding motifs and target genes in B. napus, a ChIP-Seq assay was performed. A total of 4210 and 24,578 binding peaks were identified and associated with 3939 and 15,579 neighboring genes in the CK (normal conditions as control) and DS (drought stress) sets, respectively (Supplementary Fig. 15a and Supplementary Data 5). As expected, the canonical NF-Y-binding motif CCAAT was identified in the center of the ChIP-Seq peaks, but there were significant differences in the flanking sequence of the CCAAT motif identified between CK and DS (Supplementary Fig. 15b). The target genes of BnaA9.NF-YA7 identified by ChIP-Seq were compared with the DEGs identified by RNA-Seq (Fig. 5a). In this way, common DEGs that were upregulated in BnaA9.NF-YA7-OE and downregulated in BnaA9.NF-YA7-KO or downregulated in BnaA9.NF-YA7-OE and upregulated in BnaA9.NF-YA7-KO were then extracted as high-confidence genes regulated by BnaA9.NF-YA7. A total of 1004 and 951 genes from the intersection of the Venn diagram were enriched in alpha-linolenic acid metabolism, biosynthesis of secondary metabolites, plant hormone signal transduction, and MAPK signaling pathway-plant (Supplementary Fig. 15c).

**Fig. 5: *BnaA9.NF-YA7* directly targets *BnaABF3s*.**

BnaA9.NF-YA7 directly targets BnaABF3/4s

Notably, the genes involved in plant hormone signal transduction included ABA-responsive element (ABRE)-binding factors (ABFs) (i.e., BnaABF2s, BnaABF3s, and BnaABF4s), which function redundantly as master regulators of ABA signaling in response to drought and osmotic stresses²⁰, and these genes were downregulated in the BnaA9.NF-YA7-OE lines, especially BnaABF3s and BnaC1.ABF4 (Supplementary Data 6). BnaAn.ABF3, BnaC1.ABF3, BnaC7.ABF3, and BnaC1.ABF4 were determined to have two to three potential binding sites containing CCAAT-box motifs in the promoter and coding regions (Fig. 5b and Supplementary Fig. 16a). In general, the ChIP‒qPCR values were consistent with the ChIP-Seq results (Supplementary Fig. 16b). Y2H and BiFC assays were performed to identify the BnaNF-YB/YC dimer that can interact with BnaA9.NF-YA7 to form a trimer (Supplementary Fig. 17a, b), and each of these dimers were individually fused to GAL4 DNA-BD in a Y3H system to assess their potential interaction with the BnaA9.NF-YA7 protein (Supplementary Fig. 17c, d). Y3H, α-galactosidase activity, and FRET (fluorescence resonance energy transfer) assays showed that BnaA9.NF-YB2 and BnaA6.NF-YC4 could differentially form a trimer complex with BnaA9.NF-YA7 in Hap3 (representative BnaA9.NF-YA7^G) compared to that in Hap4 (representative BnaA9.NF-YA7^C) (Supplementary Fig. 18a–c). Both the GST pull-down and co-immunoprecipitation (Co-IP) assays showed that there were interactions among BnaA9.NF-YA7, BnaA9.NF-YB2, and BnaA6.NF-YC4 in vitro and in vivo (Fig. 5c, d). EMSA also revealed band shifts when BnaA9.NF-YA7, BnaA9.NF-YB2, and BnaA6.NF-YC4 were all present (Fig. 5e and Supplementary Fig. 18d). Interestingly, the dual-luciferase reporter assay showed that neither BnaA9.NF-YA7^G- nor BnaA9.NF-YA7^C-related trimers significantly induced the LUC activity of Bna.ABF3/4 reporters (Supplementary Fig. 18e). Therefore, we speculated that BnaA9.NF-YA7-related trimers participate in Bna.ABF3/4-mediated regulatory pathways in an unknown way. To test this hypothesis, we examined the expression of downstream genes of BnaABF3s and BnaC1.ABF4 in WT, BnaA9.NF-YA7-OE, and BnaA9.NF-YA7-KO. RT‒qPCR analysis showed that the transcript levels of these downstream genes were significantly downregulated in BnaA9.NF-YA7-OE and slightly upregulated in BnaA9.NF-YA7-KO compared to WT under drought stress (Supplementary Fig. 19). These results suggest that BnaA9.NF-YA7 represses the BnaABF3/4-regulated pathway through trimerization in response to drought stress.

Next, we further analyzed the downstream target gene BnaC7.ABF3, which is an upstream regulatory factor of BnaA9.NF-YA7. In comparison with the WT, overexpression of BnaC7.ABF3 alone significantly increased survival because of increased leaf water holding capacity and decreased transpiration and water loss rates (Fig. 5f–h and Supplementary Fig. 20). In contrast, RNAi silencing of BnaC7.ABF3, led to a similar phenotype as that of the BnaA9.NF-YA7-OE lines that showed increased sensitivity to drought stress, while co-overexpression of BnaA9.NF-YA7 and BnaC7.ABF3 led to an intermediate phenotype (Fig. 5f and Supplementary Fig. 20). These results suggest that there may be a negative feedback regulatory loop between BnaA9.NF-YA7 and BnaC7.ABF3.

BnaA9.NF-YA7 regulates drought responses via regulation of the H3K36me3 status

Previous studies have established a correlation between NF-Y binding and posttranslational modifications, particularly histone modifications^{16,21,22,23,24}. To examine the specific methyltransferase activities of BnaA9.NF-YA7, the histone modification status was detected using specific antibodies through western blotting, and H3K36me3 levels were found to be specifically increased in the knockout lines and reduced in the overexpression transgenic lines compared with those of the WT (Fig. 6a, b). RNA-Seq and RT-qPCR showed that BnaA9.NF-YA7 could repress Bna.ASHH4s expression, which encodes a histone-lysine N-methyltransferase involved in histone H3K36 methyltransferase activity (Fig. 6c). Correspondingly, ChIP analysis of H3K36 trimethylation at the BnaC1.ABF3 and BnaC7.ABF3 loci in different genotypes revealed that overexpression of BnaA9.NF-YA7 significantly reduced the extent of H3K36 trimethylation at BnaC1.ABF3 and BnaC7.ABF3 (Fig. 6d, e). Comparable changes were not observed in chromatin at the BnaA2.ACT7 locus, which was used as a negative control (Supplementary Fig. 21a). Next, we performed virus-induced Bna.ASHH4s silencing using a tobacco rattle virus (TRV)-based system to reduce Bna.ASHH4s mRNA levels and determined the expression level of BnaC1.ABF3 and BnaC7.ABF3 in TRV2-Bna.ASHH4 plants. The results indicated that BnaC1.ABF3 and BnaC7.ABF3 expression were positively correlated with Bna.ASHH4s expression. The expression of BnaC1.ABF3 and BnaC7.ABF3 in TRV2-Bna.ASHH4 plants did not significantly increase even under dehydration conditions (Supplementary Fig. 21b). In turn, H3K36 trimethylation at BnaC1.ABF3 and BnaC7.ABF3 showed significant reduction in the TRV2-Bna.ASHH4 lines (Supplementary Fig. 21c). The expression level of BnaA9.NF-YA7 was not affected by the downregulation of Bna.ASHH4s under normal conditions. Moreover, ChIP analysis of H3K36 trimethylation in Hap3 and Hap4 genotypes revealed that high expression level of BnaA9.NF-YA7 significantly reduced the extent of H3K36 trimethylation at BnaC1.ABF3 and BnaC7.ABF3 loci but not BnaA4.DOR and BnaA2.ACT7 loci (Supplementary Fig. 22). Taken together, these results indicate that BnaA9.NF-YA7 suppresses the expression of BnaABF3s by indirect regulation by H3K36me3 via Bna.ASHH4s.

**Fig. 6: *BnaA9.NF-YA7* regulates drought responses via regulation of the histone H3 lysine 36 trimethylation (H3K36me3) status of the *BnaABF3s* locus.**

BnaA9.NF-YA7 inhibits ABA-induced stomatal closure via upregulating BnaA4.DOR expression

Among these common DEGs, BnaA4.DOR, which encodes the F-box protein DOR that functions as an inhibitory factor for ABA-induced stomatal closure under drought stress^25,26, was significantly upregulated in the BnaA9.NF-YA7-OE lines but barely expressed in the other genotypes (Supplementary Fig. 19). ChIP-Seq and EMSA showed that BnaA9.NF-YA7 could bind the BnaA4.DOR promoter in vivo and in vitro (Fig. 7a, b). However, ChIP analysis of H3K36 trimethylation at the BnaA4.DOR locus in different genotypes showed no significant change in the extent of H3K36 trimethylation at BnaA4.DOR (Supplementary Figs. 21a and 22). The expression level of BnaA4.DOR in Hap3 was significantly higher than that in Hap4 (Fig. 7c). Transient expression assays confirmed that co-expression of BnaA9.NF-YA7^G with the NF-YB/C dimer induced higher LUC activity of the pBnaA4.DOR reporter than co-expression of BnaA9.NF-YA7^C with NF-YB/C dimer (Fig. 7d). To further determine the role of BnaA4.DOR in BnaA9.NF-YA7-OE mediated drought sensitivity, we performed virus-induced BnaA4.DOR silencing using a TRV-based system to reduce BnaA4.DOR mRNA levels in BnaA9.NF-YA7-OE leaves (Fig. 7e). Most TRV2-BnaA4.DOR plants survived after a subsequent 5-day recovery by watering. Although the survival rate was still lower than that of the WT, it was much higher than that of BnaA9.NF-YA7-OE plants (Fig. 7e, f). The water loss rate was significantly lower in BnaA4.DOR-silenced plants than in control plants (Fig. 7g). Furthermore, the percentage of closed stomata was significantly higher in TRV2-BnaA4.DOR plants (46.80%) compared to control plants (30.75%) (Fig. 7h) and exhibiting reduced stomatal conductance and a lower transpiration rate under nonstress conditions (Fig. 7i, j). These results may explain why overexpression of BnaA9.NF-YA7 increases ABA sensitivity but does not significantly induce stomatal closure.

**Fig. 7: *BnaA9.NF-YA7* induces *BnaA4.DOR* under drought stress, thereby increasing the rate of water loss.**

Discussion

Natural variation in functional genes play extremely important roles in crop trait divergence. In rice, two SNPs in the coding region of NRT1.1B contribute to higher nitrogen-use efficiency and improved grain yield²⁷. A tandem repeat of the CCATTC sequence in the OsSPL13 5′ UTR causes reduced expression levels of OsSPL13 and results in small grains²⁸. In maize, a 366-bp insertion in the promoter of ZmVPP1 contributes significantly to drought tolerance²⁹. A two-base sequence polymorphism in UPA2 was associated with the different binding affinities of DRL1, and the wild-type UPA2 allele showed narrow leaf angles and promoted yield under high-density planting in maize³⁰. Natural variation in ZmFBL41 confers resistance to Rhizoctonia solani, that two amino acid substitutions prevent its interaction with ZmCAD, resulting in the inhibition of ZmCAD degradation and, consequently, the accumulation of lignin and the restriction of lesion expansion³¹. In cucumber, Che et al. identified a nonsynonymous polymorphism (G to A) in CsCRC as underlying the major effect of fruit size³². They found that CsCRC^G, but not CsCRC^A, targets the downstream auxin-responsive protein gene CsARP1 to regulate its expression³². These reports have confirmed that natural variations and their favorable alleles can be utilized as important genetic resources for trait improvement in crop breeding. Here, we found that a nonsynonymous polymorphism (G to C, M63I) and deletion of the CCAAT-box of the promoter in BnaA9.NF-YA7 affects the drought tolerance of B. napus. The accessions with the BnaA9.NF-YA7^C allele had a higher WHC than the accessions with the BnaA9.NF-YA7^G allele under drought treatment. The materials carrying the C allele were further divided into two types (Hap1 and Hap4), and Hap4 had stronger drought tolerance than Hap1 (Supplementary Fig. 5c). However, deletion of the CCAAT-box, which reduced the BnaA9.NF-YA7 expression level, occurred only in Hap4, suggesting that the M63I substitution cooperates with CCAAT-box deletion and can be used to increase the drought tolerance of B. napus.

Crops, including B. napus, exist in constantly changing environments and are often exposed to large amounts of abiotic and biotic stresses during their lifecycle. Among these stresses, drought stress is one of major environmental factors affecting the geographical distribution of plants in nature, limiting crop productivity in agriculture, and threatening food security³³. The ABA signaling pathway is central to drought- and salt-stress responses in plants³⁴. ABFs are known as master regulators of ABA signaling in response to drought and osmotic stresses, and the activation of genes containing an ABA-responsive element (ABRE) is largely dependent on ABF transcription factors^20,35,36. In the presence of ABA, the activation of SnRK2s leads to the phosphorylation of ABF proteins, thus allowing ABFs to induce the expression of other ABA-responsive genes^20,37. In this study, we found that fluridone (FLU) inhibited the upregulated expression of BnaA9.NF-YA7 induced by ABA treatment (Supplementary Fig. 5f), and ABFs directly regulated BnaA9.NF-YA7 expression (Fig. 4), suggesting that BnaA9.NF-YA7 may also mediate the regulation of the drought response via an ABA-dependent pathway. Under stress conditions, the activation of ABA signaling helps plants respond to environmental stress and thus help them survive under adverse conditions. However, overexpression of drought-responsive genes often results in growth deficits and yield loss³⁸. Therefore, balancing ABA signaling is very important for plants’ ability to cope with the changing environment³⁹. Studies on switching off ABA signaling have mostly focused on the genes of the PYL-PP2C-SnRK2 pathway, which is the core pathway of ABA signal transduction, and few reports have focused on ABFs and their downstream genes. In this study, we found that the NF-Y complex involving BnaA9.NF-YA7 specifically binds to the promoter and CDS of BnaABF3s and reduces histone H3 Lys 36 (H3K36) trimethylation levels to inhibit their expression, thus attenuating ABA signaling (Figs. 5 and 6). These results suggest that the negative feedback regulatory network mediated by BnaA9.NF-YA7 serves as a supplementary mechanism for ABA signal balance.

Stomatal closure preserves water in the plant, and ABA has a role in the regulation of stomatal movement to prevent water loss⁴⁰. Because stomatal movements control CO₂ influx and transpiration, drought tolerance strategies to reduce water loss via stomatal closure occur at the expense of photosynthesis, growth, and yield³⁸. Here, we found that overexpression of BnaA9.NF-YA7 increased ABA sensitivity, but the percentage of closed stomata was not significantly affected by ABA (Fig 21-q). We propose that this strategy may be part of an ABA signaling balance mechanism. BnaA9.NF-YA7 and its NF-Y complexes directly interact with the BnaA4.DOR promoter upregulates its expression level, thereby maintaining partial stomatal opening under drought stress and ensuring the CO₂ influx needed for plant photosynthesis, allowing plants to continue to grow under drought stress conditions. The M63I substitution occurred in a Pro-rich transcriptional activation domain, resulting in a transcriptional activation difference between BnaA9.NF-YA7^G and BnaA9.NF-YA7^C (Fig. 7d), thus breaking the fine-tuning of the balance between opening and closing stomata under drought stress. Nevertheless, the impact of the M63I mutation on drought tolerance is limited and only when accompanied by the CCAAT mutation can it further enhance drought tolerance. This may also explain why Hap4 exhibits stronger drought tolerance than Hap1 (Fig. 1c, d). Therefore, we posit that these two mutations are both independent yet intricately linked, with CCAAT primarily influencing the feedback regulation loop, while M63I predominantly modulates stoma sensitivity toward ABA. The excessive expression of BnaA9.NF-YA7^G/C excessively inhibited the transmission of ABA signals, and although the overexpression of BnaA9.NF-YA7^C promotes stomatal closure, it is insufficient to reverse the drought sensitivity caused by the inhibition of ABA signaling (Fig. 2f). Taken together, the data indicate that BnaA9.NF-YA7 plays a dual role as both an inhibitor and activator, effectively regulating ABA signaling in rapeseed to maintain a balance between growth and drought stress responses.

In summary, we propose the following hypothesis for how the drought tolerance difference between Hap4 and other haplotypes is regulated (Fig. 8): the expression of ABF genes and the accumulation of ABF proteins are induced by ABA signaling under drought stress, thus allowing ABFs to induce the expression of BnaA9.NF-YA7 and other ABA-responsive genes. The upregulated BnaA9.NF-YA7 can regulate the cascade strength of ABA signaling by reducing the H3K36me3 level to indirectly inhibit the expression of BnaABF3/4s, and thus BnaABF3/4s and BnaA9.NF-YA7 forms a regulatory loop to prevent excessive amplification of ABA signaling, thereby maintaining the dynamic balance between growth and the stress response in B. napus. Compared with the other haplotypes, Hap4, containing a two-base sequence variant (CCAAT-box deletion) at the promoter, is associated with a lower binding affinity for NF-Y. Low BnaA9.NF-YA7 expression in Hap4 and differences in transcriptional activation produced by the M63I substitution result in higher BnaABF3/4 s levels and a lower BnaA4.DOR level, consequently decreasing the transpiration rate and water loss rate and increasing the leaf water holding capacity. Although the contribution of BnaA9.NF-YA7 alone to improve B. napus drought tolerance was limited, our findings provide prospective drought tolerance allele. Furthermore, we add insights into the mechanisms that control ABA signaling homeostasis.

**Fig. 8: Working model demonstrating how the drought tolerance difference between Hap4 and other haplotypes is regulated at the molecular level.**

Methods

Plant materials

The diversity panel used in this study was from a previous report¹⁸. The B. napus transgenic lines used in this study were in the Zhongshuang 11 (ZS 11, Hap3) genetic background. The ProBnaA9.NF-YA7:GUS transgenic line harboring a 1697 bp BnaA9.NF-YA7 promoter fragment was transformed into Arabidopsis (Col-0) by floral dipping.

Association analysis

A total of 205 overlapping lines from previous reports of Tan et al. and Lu et al. were used for association analysis of WHC^17,18. The GWAS was conducted using the mixed linear model (MLM)⁴¹ with a total of 581,532 SNPs (miss data <20%, MAF >0.05) in the GAPIT R package⁴². The Manhattan plot was displayed using the CMplot R package⁴³.

Phylogenetic analysis of BnaA9.NF-YA7

The full-length amino acid sequence of BnaA9.NF-YA7 was used for a BLAST search of the NCBI database (http://www.ncbi.nlm.nih.gov/) to identify homologous genes in Brassicae species. The amino acid sequences were aligned using ClustalX 2.1 software. A phylogenetic tree was constructed using the neighbor-joining method with bootstrapping (1000 replicates) in MEGA version 5⁴⁴.

Plasmid construction and B. napus transformation

The BnaA9.NF-YA7 overexpression construct was generated with pENTR™ Directional TOPO® Cloning Kits (Invitrogen, USA) according to the manufacturer’s protocols, and the relevant fragment was cloned and inserted into the binary vector pEarleyGate 101. For BnaC7.ABF3 overexpression, the full-length CDS of BnaC7.ABF3 without the stop codon was amplified and cloned between the BamHI and SalI enzyme sites of the expression vector to generate the 35S:BnaC7.ABF3-DsRed fusion plasmid. The recombinant plasmids were introduced into the B. napus cultivar ZS 11 through Agrobacterium-mediated transformation. The transgenic plants were verified by PCR, and the expression levels in the transgenic plants were confirmed by RT-qPCR.

For BnaA9.NF-YA7 knockout, two 18-bp targeting sequences were selected within the BnaA9.NF-YA7 and BnaC5.NF-YA7 gene. The sequences were synthesized and inserted into the p2×35S-dpCas9-atU6-Grna-35S-Hy vector to produce the CRISPR‒Cas9 plasmid. The CRISPR‒Cas9 plasmid was introduced into the B. napus cultivar ZS 11 through Agrobacterium-mediated transformation. The target was amplified by PCR using primer pairs flanking the designed target site, and the PCR product was sequenced.

For the RNA interference (RNAi) construct, a unique 187-bp fragment from the cDNA of BnaA9.NF-YA7 was amplified and cloned between the BamHI and XbaI enzyme sites of the pFGC5941M vector, and the antisense fragment was cloned into between the NcoI and AatII enzyme sites of the pFGC5941M vector to form a hairpin structure. A unique 191-bp fragment from the cDNA of BnaC7.ABF3 was amplified and cloned into the pFGC5941M vector. The recombinant plasmids were introduced into the B. napus cultivar ZS 11 through Agrobacterium-mediated transformation. The transgenic plants were verified by PCR, and the expression levels in the transgenic plants were confirmed by RT-qPCR.

For VIGS (virus-induced gene silencing), a unique 331-bp fragment from the cDNA of BnaA4.DOR and a unique 212-bp fragment from the cDNA of Bna.ASHH4 were amplified and reverse-cloned between the BamHI and EcoRI enzyme sites of the pTRV2 vector, respectively. pTRV1 and pTRV2 were transformed into Agrobacterium tumefaciens (strain GV3101) and subsequently used for the infection of B. napus seedlings.

For site-directed mutagenesis, the site-directed mutation of the 189th base of Hap3 (G to C) was achieved by overlap extension PCR, followed by cloning the relevant fragment into the binary vector pCAMBIA1302 to create pBnaA9.NF-YA7^H3:Hap3, pBnaA9.NF-YA7^H4:Hap4, and pBnaA9.NF-YA7^H3:Hap3^C constructs, respectively. Transgenic Arabidopsis plants were generated through floral-dip by Agrobacterium-mediated transformation.

Stress treatment and physiological experiments

To assess the drought stress tolerance of WT and transgenic plants, 7-day-old seedlings were transferred to pots, grown for 3 weeks under normal conditions, and subjected to drought conditions by withholding water from the seedlings for 1 week, 10 days, or 14 days⁴⁵. The drought-related phenotypes were photographed with a camera (DS126611, Canon). The leaf water loss rate and relative water content (RWC) were determined as previously described by refs. ^8,46. Briefly, at least five leaves from different plants of each genotype were detached from 3-week-old plants and placed in a petri dish on a laboratory bench (22 ± 1 °C and 30–40% RH), and the weight of the detached leaves was then measured every 1 h for a period of 48 or 12 h. The experiment was repeated three times. Water loss was presented as the percentage of initial water loss. For the RWC, the leaves were detached from 3-week-old plants and weighed to obtain leaves weight (W), after which the leaves are immediately hydrated to full turgidity for 12 h under normal room light and temperature. After hydration, the leaves are weighed to obtain fully turgid weight (TW). Leaves are then oven-dried at 85 °C for 24 h and weighed to determine dry weight (DW). The relative water content was calculated using the following formula:

$${{{{{\rm{RWC}}}}}}(\%)=[({{{{{\rm{W}}}}}}-{{{{{\rm{DW}}}}}})/({{{{{\rm{TW}}}}}}-{{{{{\rm{DW}}}}}})]\times 100$$

(1)

To measure water holding capacity (WHC), fully expanded fourth leaves were detached from the seedlings, and the petioles were wrapped with fresh-keeping film to prevent water loss from the petioles. The fresh weight of detachment leaves was measured immediately detached from the plants (W_f), 24 h dark treatment (23 ± 1 °C and 30–40% RH, W_t), and drying (W_d). The water holding capacity of separated leaves was calculated using the following formula shown as percentage:

$${{{{{\rm{WHC}}}}}}(\%)=({{{{{{\rm{W}}}}}}}_{{{{{{\rm{t}}}}}}}-{{{{{{\rm{W}}}}}}}_{{{{{{\rm{d}}}}}}})/({{{{{{\rm{W}}}}}}}_{{{{{{\rm{f}}}}}}}-{{{{{{\rm{W}}}}}}}_{{{{{{\rm{d}}}}}}})\times 100$$

(2)

The experiment was repeated three times. For each repeat, at least five leaves from different plants of each genotype were used. The soluble protein content was determined by the Coomassie Brilliant Blue G250 method, and the free proline content was measured by the sulfosalicylic acid colorimetric method^47,48. For the stomatal aperture assay, epidermal strips were peeled from 4-week-old plants, and the mesophyll cells were removed with a small brush⁴⁵, For the ABA-mediated stomatal closure assay, the leaves were incubated in MES buffer (10 mM MES-KOH, pH 6.15, 10 mM KCl, and 50 mM CaCl₂) in light for 3 h at 22 °C to open the stomata. After the addition of 20 mM ABA or DMSO (control) to the buffer and incubation of the leaves for an additional 2 h, images were taken by an optical microscope equipped with a digital camera (Nikon ECLIPSE E600, Japan). The stomatal aperture was analyzed using ImageJ software, and three independent repeats were performed.

Histochemical GUS staining and GUS activity

The promoter sequence of BnaA9.NF-YA7 was amplified from ZS 11 (Hap3) and R219 (Hap4) and inserted into the binary vector pCAMBIA1305.1 to create pBnaA9.NF-YA7^Hap3:GUS and pBnaA9.NF-YA7^Hap4:GUS reporter gene constructs, respectively. For promoter truncation, differently truncated promoters of BnaA9.NF-YA7 were amplified and cloned between the XbaI and NcoI enzyme sites of the pCAMBIA1305.1 vector to generate promoter:GUS fusion plasmids. The recombinant plasmids were transformed into Arabidopsis (Col-0) by floral dipping. For the positive lines, plants from the T3 generation were used for GUS staining, which was performed according to the method described in the manufacturer’s protocols for the GUS Staining Kit (Coolaber, China). The stained tissues were photographed by an optical microscope equipped with a digital camera (Nikon SMZ1500, Japan). GUS activity was measured by the fluorometry method⁴⁹.

RNA extraction, qRT–PCR, and RNA-sequencing

Total RNA was extracted from 4-week-old seedlings before and after drought treatment with an EZ-10 DNAaway RNA Mini-Prep Kit (Sangon Biotech, China) according to the manufacturer’s protocols. For RT‒qPCR, cDNA was synthesized from the total RNA using a PrimeScript^TM RT reagent kit with gDNA Eraser (TaKaRa, Japan) as described in the supplier’s instructions. RT‒qPCR analyses were performed with TB Green® Premix Ex Taq™ II (Takara) on a CFX96 Real-Time PCR Detection System. PCR amplification was performed with a 3 min denaturing step at 95 °C, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. Three independent biological replicates were collected for each sample. Bna.Actin7 was used as the internal control. The comparative CT (2^-ΔCT) method⁵⁰ was used to quantify the relative gene expression levels.

For RNA-Seq, total RNA was extracted from B. napus leaves of Hap3, Hap4, WT, BnaA9.NF-YA7-OE, and BnaA9.NF-YA7-KO lines before and after drought treatment. High-quality RNA was sent to Biomarker Technologies Co., Ltd. (Beijing, China) for sequencing and analysis. TopHat2 software was used for reference genome (Darmor-bzh v4.1) mapping. Visualize the alignment results of Reads on the genome using IGV software. Transcript abundance was calculated as fragments per kilobase of transcript per million mapped reads (FPKM) values. Differentially expressed genes (DEGs) of two conditions/groups was performed using the DESeq2. The resulting P values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 found by DESeq2 were assigned as differentially expressed. The FDR <0.05 and fold change ≥1.5 was set as the threshold for significantly differential expression.

Thermal imaging

Thermal imaging of plants was performed as described previously with some modifications⁴⁵. Briefly, the plants were grown at 20 to 22 °C under a 16-h-light/8-h-dark cycle in a greenhouse for 4 weeks. Water was then withheld for 1 week before measurements. Thermal images were obtained using a WIC640 infrared camera (Workswell, Czech Republic). Leaf temperature was measured using Workswell CorePlayer professional software. For each genotype, plants in three different pots were analyzed, with similar results.

Yeast one-hybrid, two-hybrid, and three-hybrid assays

An approximately 0.5 kb core promoter DNA fragment of BnaA9.NF-YA7 was inserted upstream of the AbA^r reporter gene in the pAbAi vector as the pBait-AbAi plasmid. A B. napus cDNA library was prepared in the pGADT7-Rec vector in the Matchmaker® Gold Yeast One-Hybrid Library Screening System (PT4087-1, Clontech, USA) using RNA isolated from 4-week-old seedlings after drought stress treatment according to the manufacturer’s instructions. Yeast one-hybrid screening and one-hybrid interaction assays were performed by the user manual supplied with the Matchmaker Gold Yeast One-Hybrid Library Screening System (PT4087-1, Clontech, USA).

For the yeast two-hybrid assay (Y2H), the full-length BnaA9.NF-YA7 coding sequence was cloned and inserted into the EcoRI and BamHI sites of the prey vector pGADT7 or into the EcoRI and BamHI sites of the bait vector pGBKT7. The full-length CDSs of BnaNF-YB genes and BnaNF-YC genes were cloned and inserted into the pGADT7 and pGBKT7 vectors, respectively. The coding region of BnaC7.ABF3 was amplified and inserted into pGADT7 as AD-BnaC7.ABF3 and BnaA3.ABF4 was amplified and inserted into pGBKT7 as BD-BnaA3.ABF4. For interaction assays, the specific bait and prey constructs were co-transformed into yeast Y2HGold cells using the Yeastmaker^TM Yeast Transformation System 2 (PT1172-1, Clontech, USA) according to the manufacturer’s instructions and grown on SD/-Trp/-Leu and SD/Trp/-Leu/-His/-Ade media for interaction tests.

For the yeast three-hybrid assay (Y3H), the pBridge vector was used to express both BnaNF-YBs and BnaNF-YCs. The pGADT7 vector was used to express BnaNF-YAs, BnaC7.ABF3, and BnaA3.ABF4. Moreover, the BnaA9.NF-YB2 coding sequence was cloned and inserted into the EcoRI and BamHI sites of multiple cloning site I (MCSI) and the BnaA6.NF-YC4 coding sequence was cloned and inserted into the NotI and BglII sites of MCSII of the pBridge vector as a bait plasmid. A B. napus cDNA library was prepared in the pGADT7-Rec vector in the Matchmaker® Gold Yeast Two-Hybrid Library Screening System (PT4084-1, Clontech, USA) using RNA isolated from 4-week-old seedlings after drought stress treatment according to the manufacturer’s instructions. Yeast three-hybrid screening and three-hybrid interaction assays were performed by the user manual supplied with the Matchmaker® Gold Yeast Two-Hybrid Library Screening System (PT4084-1, Clontech, USA).

BiFC assay

The coding regions of BnaNF-Ys were amplified and inserted into the pXY104 vector containing the C-terminus of yellow fluorescent protein (cYFP) and the pXY106 vector containing the N-terminus of yellow fluorescent protein (nYFP). The coding region of BnaC7.ABF3 was amplified and inserted into the pXY104 vector and the coding region of BnaA3.ABF4 was amplified and inserted into the pXY106 vector. The plasmids were then transformed into Agrobacterium GV3101 cells with the silencing suppressor P19 strain. Then, these strains were resuspended in an induction medium (10 mM MES-KOH, pH 5.7, 10 mM MgCl₂, 200 mM acetosyringone) to an OD₆₀₀ of 1.0. After incubation at room temperature for 3 h in darkness, the different strain combinations were mixed and infiltrated into Nicotiana benthamiana leaves. The plants were grown at 22 °C under a 12-h-light/12-h-dark cycle in a greenhouse for 48 to 72 h, and YFP signals were observed under a confocal laser-scanning microscope (Zeiss LSM 800).

BiFC-FRET analysis

For BiFC-based FRET analysis, the coding regions of BnaA9.NF-YB2 and BnaA6.NF-YC4 were cloned and inserted into the pXY106 vector and pXY104 vector to produce nYFP-BnaA9.NF-YB2 and cYFP-BnaA6.NF-YC4, respectively. The full-length sequences of BnaA9.NF-YA7^G and BnaA9.NF-YA7^C were amplified, and the N-terminus was fused to the CFP coding sequence, generating BnaA9.NF-YA7^G-CFP and BnaA9.NF-YA7^C-CFP, respectively. The plasmids were then transformed into Agrobacteria GV3101 cells with the silencing suppressor P19 strain. Then, Agrobacteria cultures containing equal amounts of nYFP-BnaA9.NF-YB2 and cYFP-BnaA6.NF-YC4 strains were mixed and infiltrated into Nicotiana benthamiana leaves to allow the formation of BiFC complexes (acceptors), along with equal amounts of BnaA9.NF-YA7^G-CFP or BnaA9.NF-YA7^C-CFP (donors) strain cultures. After infiltration, the plants were grown at 22 °C under a 12-h light/12-h dark cycle in a greenhouse for 48 to 72 h before analyses of interaction using FRET. An excitation wavelength of 458 nm and an emission wavelength of 470–500 nm was used for CFP, and an excitation wavelength of 514 nm and an emission wavelength of 515–545 nm were used for YFP. The FRET energy transfer efficiency was calculated using the following formula shown as percentage^51,52:

$${{{{{\rm{E}}}}}}=({{{{{\rm{donor}}}}}}\,{{{{{\rm{after}}}}}}{{{{{\rm{\hbox{-}}}}}}}{{{{{\rm{donor\; before}}}}}})\times 100/{{{{{\rm{donor\; after}}}}}}$$

(3)

Pull-down assay

The coding regions of BnaA9.NF-YB2-Myc, BnaA6.NF-YC4-His, BnaC7.ABF3, BnaA3.ABF4, BnaA9.NF-YA7^G, and BnaA9.NF-YA7^C were cloned and inserted into the pGEX-4T-1 vector to produce GST-BnaA9.NF-YB2-Myc, GST-BnaA6.NF-YC4-His, GST-BnaC7.ABF3, GST-BnaA3.ABF4, GST-BnaA9.NF-YA7^G, and GST-BnaA9.NF-YA7^C, respectively. The expression of GST recombinant proteins in E. coli Rosetta cells (DE3; TransGen Biotech, Beijing, China) was induced by treatment with 0.5 mM IPTG for 12 h. The proteins were purified using glutathione agarose beads (Beyotime Biotechnology, Beijing, China). The BnaA9.NF-YB2-Myc and BnaA6.NF-YC4-His proteins were obtained after the GST label was digested by thrombin following the manufacturer’s instructions.

For the pull-down assay, equal amounts of BnaA9.NF-YB2-Myc and BnaA6.NF-YC4-His were incubated in binding buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and 1 mM EDTA) with immobilized GST or GST-labeled proteins at 4°C for 4 h. The Glutathione agarose beads were then mixed with the protein mixture in 1 mL of PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 1.8 mM KH₂PO₄) and mixed by rotation at 4 °C for 1 h. After washing three times with PBS, proteins that were retained on the beads were separated by SDS–polyacrylamide gel electrophoresis (PAGE). Western blot analysis was performed using anti-GST (1:2000), anti-Myc (1: 2000), and anti-His (1:2000) antibodies (Beyotime Biotechnology, Beijing, China).

Co-IP assay

The recombinant vectors (containing Myc-BnaA9.NF-YB2, His-BnaA6.NF-YC4, Flag-BnaC7.ABF3, Flag-BnaA3.ABF4, Flag-BnaA9.NF-YA7^G, and Flag-BnaA9.NF-YA7^C, respectively) were transformed into Agrobacterium tumefaciens strain GV3101 carrying p19 (35 S:p19). Then, these strains were resuspended in an induction medium to an OD₆₀₀ of 0.8. After incubation at room temperature for 3 h in darkness, the different strain combinations were mixed and infiltrated into N. benthamiana leaves. Total proteins were extracted from the samples with extraction buffer (50 mM Tris-MES (pH 8.0), 0.5 M sucrose, 1 mM MgCl₂, 10 mM EDTA, 5 mM DTT, 1 mM PMSF, and 100× protease inhibitor cocktail) at 3 d.p.i and incubated with anti-Flag antibody immobilized onto Protein G/A-Agarose suspension (Beyotime Biotechnology, Beijing, China) in extraction buffer at 4 °C for 4 h. After incubation, the beads were washed with wash buffer (100 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1% [v/v] Triton X-100) and eluted with 5× SDS loading buffer. The proteins bound to the beads were resolved by SDS–PAGE and detected by anti-FLAG, anti-Myc, and anti-His antibodies (Beyotime Biotechnology, Beijing, China).

Transient expression assay

For the dual-luciferase transcriptional activity assays, the firefly luciferase (LUC) gene driven by the minimal TATA box of the CaMV 35S promoter following five copies of the GAL4 binding element was used as a reporter⁵³. BnaA9.NF-YA7 CDS fragments of different lengths fused with the yeast GAL4 DNA-binding domain driven by the CaMV 35S promoter followed by the translational enhancer from tobacco mosaic virus were used as effectors⁵⁴.

For the dual-luciferase transient expression assays, the different promoter segments were recombined into the HindIII and BamHI sites of the pGreenII 0800-LUC vector, generating the promoter:LUC plasmids as reporters. The Renilla luciferase (REN) gene driven by the 35S promoter in the pGreenII 0800-LUC vector was used as the internal control³⁰. The full-length CDSs of different genes were amplified and recombined into the BamHI and EcoRI sites of the pGreenII 62-SK vector driven by the 35S promoter, forming different effectors. The empty pGreenII 62-SK vector was used as a control.

Transient dual-luciferase assays were performed in Arabidopsis protoplasts collected from the leaves of 4-week-old seedlings. The luciferase signal was detected using the Dual-Luciferase Reporter Assay System (Promega, USA) with a GloMax 20/20 Luminometer (Promega, USA). Relative LUC activity was calculated by normalizing LUC activity to REN activity³⁰.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assays were performed using an EpiQuik^TM Plant ChIP kit (Epigentek) according to the user guide. Leaf tissues (1 g) before and after drought treatment were collected from 35S:BnaA9.NF-YA7-GFP transgenic seedlings grown in pots for 4 weeks. An anti-GFP antibody (ab290; Abcam) was used to immunoprecipitate the sonicated DNA‒protein complexes containing BnaA9.NF-YA7-GFP. The qualified chromatin fragments were sent to Novogene (www.novogene.com/) for ChIP-Seq analysis. High-quality clean reads were mapped to the public B. napus genome Darmor-bzh v4.1 with default parameters using the Burrows–Wheeler Aligner (BWA)⁵⁵.

To determine whether BnaABF3/4 regulates and controls BnaA9.NF-YA7, chromatin immunoprecipitation was performed using BnaC7.ABF3-Flag, BnaA3.ABF4-Flag, and empty vector transgenic plant leaves. Sonicated chromatin fragments were immunoprecipitated using an anti-Flag antibody (CST, 14793S). For histone modification analysis, ChIP experiments were performed using WT, BnaA9.NF-YA7-OE, and BnaA9.NF-YA7-KO leaves. ChIP was performed using Protein A/G magnetic beads (ab193262; Abcam) and anti-H3K36me3 antibody (Abcam, ab9050), anti-H3K4me3 antibody (Abcam, ab8580), anti-H3K27me3 antibody (Abcam, ab6002), and anti-Histone H3 antibody (Abcam, ab1791). The precipitated DNA fragments were recovered and analyzed by RT‒qPCR using specific primers. ChIP‒qPCR assays were performed using three biological replicates and three technical replicates for each biological replicate. The fold enrichment was calculated against the GFP or Flag control samples.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

Sequencing data associated with this study have been deposited in the NCBI Sequence Read Archive under BioProject accessions PRJNA977421, PRJNA978264, and PRJNA978979. The primers used in this work are listed in Supplementary Table 2, Supplementary Data 7, and Supplementary Data 8. Source data are provided with this paper.

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Acknowledgements

We thank Huanhuan Jiang for providing the RNA interference vector, Xiupeng Mei (State Cultivation Base of Crop Stress Biology for Southern Mountainous Land, Southwest University) for providing the dual-luciferase transcriptional activity vector, and Lisha Zhang (Key Laboratory of Application and Safety Control of Genetically Modified Crops, Southwest University) for transient expression assays. This research was supported by the National Key Research and Development Program of China to L.L. (2022YFD1200400) and the National Natural Science Foundation of China to L.L. (31971902 and 32001509).

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College of Agronomy and Biotechnology, Southwest University, Beibei, Chongqing, 400715, China
Jia Wang, Lin Mao, Yangyang Li, Kun Lu, Cunmin Qu, Zhanglin Tang, Jiana Li & Liezhao Liu
Academy of Agricultural Science, Southwest University, Beibei, Chongqing, 400715, China
Jia Wang, Lin Mao, Yangyang Li, Kun Lu, Cunmin Qu, Zhanglin Tang, Jiana Li & Liezhao Liu

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Jia Wang
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Lin Mao
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Yangyang Li
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Kun Lu
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Cunmin Qu
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Zhanglin Tang
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Jiana Li
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Liezhao Liu
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L.L. designed the study. J.W. and L.M. performed the experiments. J.W., L.M., and Y.L. analyzed the data. C.Q., K.L., Z.T., J.L., and L.L. collected B. napus cultivars. J.W. wrote the manuscript. All authors have read and approved the final manuscript.

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Correspondence to Liezhao Liu.

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Wang, J., Mao, L., Li, Y. et al. Natural variation in BnaA9.NF-YA7 contributes to drought tolerance in Brassica napus L. Nat Commun 15, 2082 (2024). https://doi.org/10.1038/s41467-024-46271-2

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Received: 23 August 2023
Accepted: 21 February 2024
Published: 07 March 2024
DOI: https://doi.org/10.1038/s41467-024-46271-2

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