Gene editing of authentic Brassica rapa flavonol synthase 1 generates dihydroflavonol-accumulating Chinese cabbage

Flavonols are the major class of flavonoids of green Chinese cabbage(Brassica rapa subsp.pekinensis).The B.rapa genome harbors seven flavonol synthase genes(BrFLSs),but they have not been functionally characterized.Here,transcriptome analysis showed four BrFLSs mainly expressed in Chinese cabbage.Among them,only BrFLS1 showed major FLS activity and additional flavanone 3β-hydroxylase(F3H)activity,while BrFLS2 and BrFLS3.1 exhibited only marginal F3H activities.We generated BrFLS1-knockout(BrFLS1-KO)Chinese cabbages using CRISPR/Cas9-mediated genome editing and obtained transgene-free homozygous plants without off-target mutation in the T1 generation,which were further advanced to the T2 generation showing normal phenotype.UPLC-ESI-QTOF-MS analysis revealed that flavonol glycosides were dramatically decreased in the T2 plants,while dihydroflavonol glycosides accumulated concomitantly to levels corresponding to the reduced levels of flavonols.Quantitative PCR analysis revealed that the early steps of phenylpropanoid and flavonoid biosynthetic pathway were upregulated in the BrFLS1-KO plants.In accordance,total phenolic contents were slightly enhanced in the BrFLS1-KO plants,which suggests a negative role of flavonols in phenylpropanoid and flavonoid biosynthesis in Chinese cabbage.Phenotypic surveys revealed that the BrFLS1-KO Chinese cabbages showed normal head formation and reproductive phenotypes,but subtle morphological changes in their heads were observed.In addition,their seedlings were susceptible to osmotic stress compared to the controls,suggesting that flavonols play a positive role for osmotic stress tolerance in B.rapa seedling.In this study,we showed that CRISPR/Cas9-mediated BrFLS1-KO successfully generated a valuable breeding resource of Chinese cabbage with distinctive metabolic traits and that CRISPR/Cas9 can be efficiently applied in functional Chinese cabbage breeding.

CIPK9: a calcium sensor-interacting protein kinase required for low-potassium tolerance in Arabidopsis

Potassium is one of the major macro-nutrients essential for a number of cellular processes in plants. Low potassium level in the soil represents a limiting factor for crop production. Recent studies have identified potassium transporters that are involved in potassium acquisition, and some of them are critical for potassium nutrition under low potassium conditions. However, little is understood on the molecular components involved in low potassium signaling and responses. We report here the identification ofa calcineurin B-like protein-interacting protein kinase （CIPK9） as a critical regulator of low potassium response in ,Arabidopsis. The CIPK9 gene was responsive to abiotic stress conditions, and its transcript was inducible in both roots and shoots by potassium deprivation. Disruption of CIPK9 function rendered the mutant plants hypersensitive to low potassium media. Further analysis indicated that K^＋ uptake and content were not affected in the mutant plants, implying CIPK9 in the regulation of potassium utilization or sensing processes.

Calcineurin-B-Like Protein CBL9 Interacts with Target Kinase CIPK3 in the Regulation of ABA Response in Seed Germination

Calcium plays a vital role as a second messenger in many signaling pathways in plants. The calcineurin B-like proteins （CBLs） represent a family of plant calcium-binding proteins that function in calcium signaling by interacting with their interacting protein kinases （CIPKs）. In our previous study, we have reported a role for one of the CBLs （CBL9） and one of the CIPKs （CIPK3） in ABA signaling. Here, we have shown that CBL9 and CIPK3 physically and functionally interact with each other in regulating the ABA responses. The CBL9 and CIPK3 proteins interacted with each other in the yeast two- hybrid system and when expressed in plant cells. The double mutant cbl9cipk3 showed the similar hypersensitive response to ABA as observed in single mutants （cbl9 or cipk3）. The constitutively active form of CIPK3 genetically complemented the cbl9 mutant, indicating that CIPK3 function downstream of CBL9. Based on these findings, we conclude that CBL9 and CIPK3 act together in the same pathway for regulating ABA responses.

Expression Analysis of Proline Metabolism-related Genes From Halophyte Arabis stelleri under Osmotic Stress Conditions

Arabis stelleri var. japonica evidenced stronger osmotic stress tolerance than Arabidopsis thaliana. Using an A. thaliana microarray chip, we determined changes in the expression of approximately 2 800 genes between A. stelleri plants treated with 0.2 M mannitol versus mock-treated plants. The most significant changes in the gene expression patterns were in genes defining cellular components or in genes associated with the endomembrane system, stimulus response, stress response, chemical stimulus response, and defense response. The expression patterns of three de novo proline biosynthesis enzymes were evaluated in A. stelleri var. japonica seedlings treated with 0.2 M mannitol, 0.2 M sorbitol, and 0.2 M NaCI. The expression of At-pyrroline-5-carboxylate synthetase was not affected by NaCI stress but was similarly induced by mannitol and sorbitol. The proline dehydrogenase gene, which is known to be repressed by dehydration stress and induced by free L-proline, was induced at an early stage by mannitol treatment, but the level of proline dehydrogenase was increased later by treatment with both mannitol and NaCI. The level of free L-proline accumulation increased progressively in response to treatments with mannitol, sorbitol, and NaCI. Mannitol induced L-proline accumulation more rapidly than NaCI or sorbitol. These findings demonstrate that the osmotic tolerance of the novel halophyte, Arabis stelleri, is associated with the accumulation of L-proline.