The CRISPR/Cas9 technique is a highly valuable tool in creating new materials for both basic and applied researches. Previously, we succeeded in effectively generating mutations in Brassica napus using an available CRISPR/Cas9 vector pKSE401, while isolation of Cas9-free mutants is laborious and inefficient. Here, we inserted a fluorescence tag (sGFP) driven by the constitutive 35S promoter into pKSE401 to facilitate a visual screen of mutants. This modified vector was named pKSE401G and tested in several dicot plant species, including Arabidopsis, B. napus, Fragaria vesca (strawberry), and Glycine max (soybean). Consequently, GFP-positive plants were readily identified through fluorescence screening in all of these species. Among these GFP-positive plants, the average mutation frequency ranged from 20.4 to 52.5% in Arabidopsis and B. napus with stable transformation, and was 90.0% in strawberry and 75.0% in soybean with transient transformation, indicating that the editing efficiency resembles that of the original vector. Moreover, transgene-free mutants were sufficiently identified in Arabidopsis in the T2 generation and B. napus in the T1 generation based on the absence of GFP fluorescence, and these mutants were stably transmissible to next generation without newly induced mutations. Collectively, pKSE401G provides us an effective tool to readily identify positive primary transformants and transgene-free mutants in later generations in a wide range of dicot plant species.

Drought is a major threat to plant growth and crop productivity. Reduced level of the gibberellin would result in increased drought tolerance, but the underlying mechanism is still unclear. In Brassica napus, there are four BnaRGA genes that code for DELLA proteins, negative regulators of GA signaling. Among them, expression of BnaA6.RGA was greatly induced by drought and abscisic acid (ABA). Previously, we created the gainof-function mutant of BnaA6.RGA, bnaa6.rga-D, and the loss-of-function quadruple mutant, bnarga by CRISPR/Cas9, respectively. Here we show that bnaa6.rga-D displayed enhanced drought tolerance, and its stomatal closure was hypersensitive to ABA treatment. By contrast, bnarga displayed reduced drought tolerance and was less sensitive to ABA treatment, but there is no difference in drought tolerance between single BnaRGA mutant and WT, suggesting a functional redundancy between the BnaRGA genes in this process. Furthermore, we found that BnaRGAs were able to interact physically with BnaA10.ABF2, an essential transcription factor in ABA signaling. The BnaA10.ABF2-BnaA6.RGA protein complex greatly increased the expression level of the drought responsive gene BnaC9.RAB18. Taken together, this work highlighted the fundamental roles of DELLA proteins in drought tolerance in B. napus, and provide desirable germplasm for further breeding of drought tolerance in rapeseed.

Seed oil content (SOC) and fatty acid (FA) composition determine the quality and economic value of rapeseed (Brassica napus). Little is known about the role of gibberellic acid (GA) in regulating FA biosynthesis in B. napus. Here, we discovered that four BnaRGAs (B. napus REPRESSOR OF GA), encoding negative regulators of GA signalling, were suppressed during seed development. Compared to the wild type, SOC was reduced in gain-of-function mutants bnaa6.rga-D and ds-3, which also showed reduced oleic acid and increased linoleic acid contents. By contrast, the loss-of-function quadruple mutant bnarga displayed higher SOC during early seed development than the wild type, with increased oleic acid and reduced linoleic acid contents. Notably, only BnaA6.RGA and BnaC7.RGA physically interacted with two BnaLEC1s, which function as essential transcription factors in FA biosynthesis. The FA composition did not significantly differ between bnarga bnalec1 sextuple mutants and bnalec1, suggesting that BnaLEC1s are epistatic to BnaRGAs in the regulation of FA composition. Furthermore, BnaLEC1-induced activation of BnaABI3 expression was repressed by BnaA6.RGA, indicating that GA triggers the degradation of BnaRGAs to relieve their repression of BnaLEC1s, thus promoting the transcription of downstream genes to facilitate oil biosynthesis. Therefore, we uncovered a developmental stagespecific role of GA in regulating oil biosynthesis via the GA-BnaRGA-BnaLEC1 signalling cascade, providing a novel mechanistic understanding of how phytohormones regulate FA biosynthesis in seeds. BnaRGAs represent promising targets for oil crop improvement.

Plant hormones are the intrinsic factors that control plant development. The integration of different phytohormone pathways in a complex network of synergistic, antagonistic and additive interactions has been elucidated in model plants. However, the systemic level of transcriptional responses to hormone crosstalk in Brassica napus is largely unknown. Here, we present an indepth temporal-resolution study of the transcriptomes of the seven hormones in B. napus seedlings. Differentially expressed gene analysis revealed few common target genes that coregulated (up-and down-regulated) by seven hormones; instead, different hormones appear to regulate distinct members of protein families. We then constructed the regulatory networks between the seven hormones side by side, which allowed us to identify key genes and transcription factors that regulate the hormone crosstalk in B. napus. Using this dataset, we uncovered a novel crosstalk between gibberellin and cytokinin in which cytokinin homeostasis was mediated by RGA-related CKXs expression. Moreover, the modulation of gibberellin metabolism by the identified key transcription factors was confirmed in B. napus. Furthermore, all data were available online from http://yanglab.hzau.edu.cn/BnTIR/hormone. Our study reveals an integrated hormone crosstalk network in Brassica napus, which also provides a versatile resource for future hormone studies in plant species.

Cytokinins (CKs) are the phytohormones that promote cell division and differentiation. However, the regulation of CKs distribution and homeostasis in Brassica napus is poorly understood. Here, the endogenous CKs were first quantified by LC-ESI-MS/MS in rapeseed tissues and visualized by TCSn::GUS reporter lines. Interestingly, the cytokinin oxidase/dehydrogenase BnaCKX2 homologs were mainly expressed in reproductive organs. Later, the quadruple mutants of the four BnaCKX2 homologs were generated. Endogenous CKs were increased in the seeds of the BnaCKX2 quadruple mutants, resulting in a significantly reduced seed size. In contrast, overexpression of BnaA9.CKX2 resulted in larger seeds, probably by delaying endosperm cellularization. Furthermore, BnaC6.WRKY10b, but not BnaC6.WRKY10a, positively regulated BnaA9.CKX2 expression by binding directly to its promoter region. Overexpression of BnaC6.WRKY10b rather than BnaC6.WRKY10a resulted in lower CKs levels and larger seeds by activating BnaA9.CKX2 expression, indicating that the functional differentiation of BnaWRKY10 homologs might have occurred during the B. napus evolution or domestication. Notably, the haploid types of BnaA9.CKX2 were associated with 1000-seed weight in the natural B. napus population. Overall, the study reveals the distribution of CKs in B. napus tissues, and highlights that the BnaWRKY10-mediated BnaCKX2 expression is essential for seed size regulation, providing promising targets for oil crop improvement.