First report of Rhizoctonia blight caused by Rhizoctonia solani on Chinese cabbage (Brassica rapa subsp. chinensis) in India.

During January and February 2021, foliar blight symptoms were observed on the leaves of Chinese cabbage (Pak choi) at Lembucherra research farm, College of Agriculture, Tripura, India. The incidence of disease symptoms ranged from 5 to 10% of the plants observed in the field. The symptomatic leaves showed grayish colored water-soaked lesions with an irreguar shape and size. A total of 10 symptomatic leaves (1 leaf per plant) from Chinese cabbage infected plant were sampled, surface decontaminated with 1% NaOCl, washed twice in sterile water, plated on 2% water agar, and incubated at 25 ± 2°C. Hyphal tips from mycelium of 7-day old culture (2 isolates from two different plants) with right-angled branching were transferred to potato dextrose agar (PDA) media (SRL, India). Cream or light brown hyphae that branched at right angles, with septa near the point of the origin of hyphae, and a slight constriction at the base of the branch) were visible under a microscope. Olive-brown sclerotia were observed after 5 days of incubation. Multiple nuclei per cell were visible after staining with 4', 6-diamidino-2-phenylindole (Estandarte et al. 2016). Based on morphological characteristics (Parmeter et al. 1970) the isolates TP36 and TP37 were identified as Rhizoctonia solani. The internal transcribed spacer (ITS) region and glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) were amplified with ITS1& ITS4 (White et al. 1990) and (GAPDH F-5'- CAAGGAGAACCCAGGTGTTAAG-3' and GAPDH R- 5'-GGCGTCGAAGATAGAAGAGTGT-3') respectively for both isolates and sequenced (accession #. PP458158, PP458159, PP425343, PP425344). BLASTn analysis showed 99.26%( 668/673 nt) to 99.46% (659/664 nt) identity with R. solani sequences (GenBank MG397062.1 and KX674524.1) for ITS and 98.42% (552/562 nt) to 100% 540/540 nt)identity with R. solani sequences (GenBank HQ425709.1 and CP102644.1) for GAPDH. Isolates TP36 and TP37 were deposited in the Indian Type Culture Collection (ITCC), New Delhi as R. solani (nos. 9154 and 9319, respectively). Both isolates were amplified using (anastomosis group) AG1 subgroup specific primers (Matsumoto 2002; Prashantha et al. 2021) to identify their AG. The presence of a 265 bp amplicon for both isolates suggested that they belong to AG1-IA. A multilocus analysis of R. solani isolates from different host plants with concatenated sequences ITS and GAPH showed that TP36 and TP37 are closely related to rice isolate RS107. A pathogenicity test on five plants per treatment was conducted and repeated twice on one month old Chinese cabbage plants (hybrid, TOKITA, India) grown under glasshouse conditions in a sterilized mixture of soil and sand (3:1) at 27-28oC during January 2024 at ICAR-IARI, New Delhi. R. solani isolates TP36 and TP37 were grown on PDA and plants were inoculated by placing single sclerotia of 10-day old colony on different plant parts and covering it with moist cotton. After 7 day, typical lesions of R. solani infection were visible. No symptoms were observed on the control plants. The fungus was reisolated from the inoculated plants and identified as R. solani based on morphology. R. solani has previously been reported to cause disease on some members of Brassicaceae in different countries (Budge et al. 2009; Hua et al. 2014). Based on literature available this is the first report of R. solani infecting Chinese cabbage in India.

Potential Regulatory Networks and Heterosis for Flavonoid and Terpenoid Contents in Pak Choi: Metabolomic and Transcriptome Analyses.

Pak choi exhibits a diverse color range and serves as a rich source of flavonoids and terpenoids. However, the mechanisms underlying the heterosis and coordinated regulation of these compounds-particularly isorhamnetin-remain unclear. This study involved three hybrid combinations and the detection of 528 metabolites from all combinations, including 26 flavonoids and 88 terpenoids, through untargeted metabolomics. Analysis of differential metabolites indicated that the heterosis for the flavonoid and terpenoid contents was parent-dependent, and positive heterosis was observed for isorhamnetin in the two hybrid combinations (SZQ, 002 and HMG, ZMG). Moreover, there was a high transcription level of flavone 3'-O-methyltransferase, which is involved in isorhamnetin biosynthesis. The third group was considered the ideal hybrid combination for investigating the heterosis of flavonoid and terpenoid contents. Transcriptome analysis identified a total of 12,652 DEGs (TPM > 1) in various groups that were used for comparison, and DEGs encoding enzymes involved in various categories, including "carotenoid bio-synthesis" and "anthocyanin biosynthesis", were enriched in the hybrid combination (SZQ, 002). Moreover, the category of anthocyanin biosynthesis also was enriched in the hybrid combination (HMG, ZMG). The flavonoid pathway demonstrated more differential metabolites than the terpenoid pathway did. The WGCNA demonstrated notable positive correlations between the dark-green modules and many flavonoids and terpenoids. Moreover, there were 23 ERF genes in the co-expression network (r ≥ 0.90 and p < 0.05). Thus, ERF genes may play a significant role in regulating flavonoid and terpenoid biosynthesis. These findings enhance our understanding of the heterosis and coordinated regulation of flavonoid and terpenoid biosynthesis in pak choi, offering insights for genomics-based breeding improvements.

BrABF3 promotes flowering through the direct activation of CONSTANS transcription in pak choi.

Drought stress triggers the accumulation of the phytohormone abscisic acid (ABA), which in turn activates the expression of the floral integrator gene CONSTANS (CO), accelerating flowering. However, the molecular mechanism of ABA-induced CO activation remains elusive. Here, we conducted a yeast one-hybrid assay using the CO promoter from Brassica campestris (syn. Brassica rapa) ssp. chinensis (pak choi) to screen the ABA-induced pak choi library and identified the transcription activator ABF3 (BrABF3). BrABF3, the expression of which was induced by ABA in pak choi, directly bound to the CO promoter from both pak choi and Arabidopsis. The BrABF3 promoter is specifically active in the Arabidopsis leaf vascular tissue, where CO is mainly expressed. Impaired BrABF3 expression in pak choi decreased BrCO expression levels and delayed flowering, whereas ectopic expression of BrABF3 in Arabidopsis increased CO expression and induced earlier flowering under the long-day conditions. Electrophoretic mobility shift assay analysis showed that BrABF3 was enriched at the canonical ABA-responsive element-ABRE binding factor (ABRE-ABF) binding motifs of the BrCO promoter. The direct binding of BrABF3 to the ABRE elements of CO was further confirmed by chromatin immunoprecipitation quantitative PCR. In addition, the induction of BrCO transcription by BrABF3 could be repressed by BrCDF1 in the morning. Thus, our results suggest that ABA could accelerate the floral transition by directly activating BrCO transcription through BrABF3 in pak choi.

BcNAC056 Interacts with BcWRKY1 to Regulate Leaf Senescence in Pak Choi.

Senescence is the final stage of leaf development. For leafy vegetables such as pak choi, leaf senescence is adverse to yield due to the harvest period shortening. However, the regulatory mechanisms of leaf senescence are largely unknown in leafy vegetables. Here, we isolated and characterized a NAC gene, BcNAC056, in pak choi [Brassica campestris (syn. Brassica rapa) ssp. chinensis cv. 49caixin]. BcNAC056-GFP was located in the nucleus at the subcellular level, and BcNAC056 was responsive to leaf senescence and different hormones at the transcriptional level. Heterologous overexpression of BcNAC056 in Arabidopsis promoted leaf senescence, accompanied by the increased expression of senescence-associated genes (SAGs), whereas virus-induced gene silencing-based silencing in pak choi delayed leaf senescence. The following transcriptome analysis showed that heterologous overexpression of BcNAC056 enhanced some AtSAG transcripts in Arabidopsis. Electrophoretic mobility shift assay (EMSA) and dual-luciferase (LUC) reporter assay revealed that BcNAC056 activated SAG12 by directly binding to the promoter. In addition, with the LUC reporter and transient overexpression assays, we proposed that BcNAC056-BcWRKY1 interaction promoted the activation of BcSAG12. Taken together, our findings revealed a new regulatory mechanism of leaf senescence in pak choi.

Metabolic and Transcriptomic Analyses Reveal Different Metabolite Biosynthesis Profiles between Purple and Green Pak Choi.

Pak choi is one of the most important leafy vegetables planted in East Asia and provides essential nutrients for the human body. Purple pak choi differs mainly in leaf colour but exhibits distinct nutritional profiles from green pak choi. In this study, we performed metabolic and transcriptomic analyses to uncover the mechanisms underlying the differences in metabolite biosynthesis profiles between the two pak choi varieties. Metabolite profiling revealed significant differences in the levels of metabolites, mainly amino acids and their derivatives and flavonoids. Furthermore, 34 flavonoids significantly differed between green and purple pak choi leaves, and cyanidin and its derivative anthocyanins were abundant in purple pak choi. In addition, we found that the structural genes CHS, DFR, ANS, and UGT75C1, as well as the transcription factor MYB2, play a major role in anthocyanin synthesis. These results provide insight into the molecular mechanisms underlying leaf pigmentation in pak choi and offer a platform for assessing related varieties.

Effect of Plasma-Treated Water with Magnesium and Zinc on Growth of Chinese Cabbage.

Nonthermal biocompatible plasma (NBP) is an emerging technology in the field of agriculture to boost plant growth. Plasma is a source of various gaseous reactive oxygen and nitrogen species (RONS) and has a promising role in agricultural applications, as the long-lived RONS (H2O2, NO2-, NO3-) in liquid activate signaling molecules in plant metabolism. Plasma-treated water (PTW) has an acidic pH of around 3 to 4, which may be detrimental to pH-sensitive plants. Innovative techniques for producing PTW with a pH value of 6 to 7 under neutral circumstances are desperately required to broaden the application range of NBP in agriculture. Furthermore, Pak Choi (Brassica campestris L.) is a Brassicaceae family green vegetable that has yet to be investigated for its response to NBP. In this work, we proposed an alternate method for neutralizing the pH of PTW by immersing metal ions (Mg2+ and Zn2+) in the PTW and observing its effect on Pak Choi. After synthesizing PTW with MECDBD, we analyzed germination rate and growth parameters, then seedlings for 42 days to show physiological, biochemical, and molecular levels. The germination rate was observed to be higher with PTW and more efficient when metal ions were present. Seedling length and germination rates were dramatically boosted when compared to DI water irrigation. Because of the increased chlorophyll and protein content, the plants responded to the availability of nitrogen by generating highly green leaves. Furthermore, we observed that PTW increases the expression of NR genes and GLR1 genes, which are further increased when metals are submerged in the PTW. Furthermore, PTW and PTW with metals reduced ABI5 and CHO1 which is associated with a growth inhibitor. According to this study, nonthermal plasma might be utilized to significantly improve seed germination and seedlings' development.

BcWRKY1 confers salt sensitivity via inhibiting Reactive oxygen species scavenging.

WRKY transcription factors play important roles in abiotic stress by directly regulating stress-related genes. However, the molecular mechanism of its involvement in salt stress in pak-choi is still poorly understood. In this study, we elucidated the function of BcWRKY1 from pak-choi (Brassica rapa ssp. chinensis) in salt stress. The expression level of BcWRKY1 showed the highest in rosette leaves among different tissues and was induced by salt and ABA treatment in pak-choi. Subcellular localization showed that BcWRKY1 was located in nucleus. The transgenic Arabidopsis overexpressing BcWRKY1 exhibited enhanced salt sensitivity and higher H2O2 contents, which were further confirmed by silencing BcWRKY1 in pak-choi. In addition, the expression of ZAT12 was negatively regulated with BcWRKY1 under salt stress both in pak-choi and Arabidopsis. Yeast one-hybrid and dual luciferase reporter assay showed that BcWRKY1 could bind to the promoter of BcZAT12, and BcsAPX expression was activated by BcZAT12. To sum up, we propose a BcWRKY1-BcZAT12-BcsAPX regulatory model that involves in pak-choi salt stress response.

Foliar Application of Reaction Products Derived from Selenite Removal by Iron Monosulfide for Brassica rapa ssp. Chinensis L.

The extensive application of FeS in environmental remediation requires the recovery and reuse of reaction products between FeS and pollutants. Therefore, foliar application of reaction products derived from selenite [Se(IV)] removal by FeS for pak choi was performed. The removal rate of Se(IV) by 100 mg/L FeS was 0.047 h-1. 93.2% of Se(IV) was reduced to Se(0), and FeS was correspondingly oxidized to goethite (78.9%), lepidocrocite (21.1%), and S(0) (91.5%) based on the analysis of X-ray absorption fine structure. The reaction products promoted the growth of pak choi in terms of fresh biomass, vitamin C, and protein, ascribed to the key roles of Fe and S in enhancing the electron transfer rate and light conversion rate. Furthermore, the application of reaction products decreased by 64% of disease incidence as compared with the pathogen Pseudomonas syringae pv. maculicola-infected control. The total Se content in plants increased to 576 µg/kg and was composed of 11.9% of SeMeCys, 29.8% of SeMet, and 58.3% of SeCys after exposure to reaction products, which is beneficial to the human dietary intake from pak choi. This study demonstrated that the reaction products between FeS and Se(IV) could be recovered and applied as a nano-enabled strategy to prevent crop insecurity.

Enhanced photosynthetic activity in pak choi hybrids is associated with increased grana thylakoids in chloroplasts.

Increased photosynthetic activity is closely linked to heterosis in plants, but the underlying molecular mechanisms remain elusive. Pak choi (Brassica rapa ssp. chinensis) is a widely grown vegetable in Asia, and the most commercial cultivars are F1 hybrids. Here, the inbred pak choi lines WTC and 2Q, and their reciprocal F1 hybrids WQ and QW, were used to characterize the increased photosynthetic activity in these hybrids at the physiological, cellular and molecular levels. We found that the hybrids had larger leaves, with more grana thylakoids. Additionally, these hybrids had significantly increased net photosynthetic rates (Pn ) under both saturating and low irradiance conditions. These data indicate that the increased photosynthetic activity in pak choi hybrids was associated with an improved photosynthetic mechanism and larger leaves. Next, we obtained genome-wide data using transcriptome and bisulfite sequencing. Gene ontology (GO) analysis showed that the differentially expressed genes among the parents and hybrids were mostly enriched in the 'photosynthesis', 'thylakoid', and 'chloroplast' categories, indicating that the increased number of grana thylakoids contributes to the enhanced photosynthetic capacity in hybrids. Furthermore, we found that the increased number of grana thylakoids was associated with the upregulation of light-harvesting complex of photosystem II 1 (BrLhcb1). Yeast one-hybrid and transient assay showed that the BrLhcb1 promoter was directly bound by CIRCADIAN CLOCK ASSOCIATED 1 (BrCCA1), resulting in increased BrLhcb1 expression and enhanced carbon fixation in hybrids. Finally, our findings provide new insight into molecular mechanisms underlying enhanced photosynthesis in pak choi hybrids.

Assembly of the non-heading pak choi genome and comparison with the genomes of heading Chinese cabbage and the oilseed yellow sarson.

Brassica rapa displays a wide range of morphological diversity which is exploited for a variety of food crops. Here we present a high-quality genome assembly for pak choi (Brassica rapa L. subsp. chinensis), an important non-heading leafy vegetable, and comparison with the genomes of heading type Chinese cabbage and the oilseed form, yellow sarson. Gene presence-absence variation (PAV) and genomic structural variations (SV) were identified, together with single nucleotide polymorphisms (SNPs). The structure and expression of genes for leaf morphology and flowering were compared between the three morphotypes revealing candidate genes for these traits in B. rapa. The pak choi genome assembly and its comparison with other B. rapa genome assemblies provides a valuable resource for the genetic improvement of this important vegetable crop and as a model to understand the diversity of morphological variation across Brassica species.

Plasma membrane-localized protein BcHIPP16 promotes the uptake of copper and cadmium in planta.

Cadmium (Cd) is one of the toxic heavy metals in soil, which not only suppresses crop production but also threatens human health. In this study, we aim to clarify the biological function of Cd-related gene BcHIPP16, so as to provide potential genetic solutions to decrease the Cd levels of pak choi. Tissue expression analysis showed that BcHIPP16 expressed in almost all the plant bodies. The transcriptional level of BcHIPP16 in roots was higher than that in shoots, which was significantly induced by copper (Cu) deficiency and Cd exposure conditions. Subcellular localization revealed that BcHIPP16 localized in plasma membrane. Expressing BcHIPP16 in yeast cells improved the sensitivity to Cu and Cd and improved their accumulation in yeast. Furthermore, the Cu and Cd content of Arabidopsis seedlings were increased and complemented, respectively when expressing BcHIPP16 in wild type (WT) and hip16 mutants. Non-invasive Micro-test Technology (NMT) was used to measure the real-time Cd2+ influx from the root surface of BcHIPP16 transgenic Arabidopsis lines, and the result demonstrated that BcHIPP16 promoted Cd2+ influx into Arabidopsis root cells. Taken together, our study showed that BcHIPP16 contributed to absorbing nutrient metal Cu and heavy metal Cd in planta.

Residue behavior and dietary risk assessment of six pesticides in pak choi using QuEChERS method coupled with UPLC-MS/MS.

A reliable and simple modified QuEChERS method with UPLC-MS/MS was developed for the simultaneous determination of six pesticides (dimethomorph, imidaclothiz, lufenuron, methoxyfenozide, pyridaben, spinetoram) and their metabolites in pak choi. Method validation indicated good linearity (R2 ≥ 0.99), accuracy (recoveries of 75%-112%), sensitivity (limits of quantification, 0.002-0.01 mg kg-1), and precision (relative standard deviations ≤ 21%), and matrix effects were -36-28%. The half-lives of the six pesticides in pak choi were 2.2-12 d under open field and greenhouse conditions. Considering the short growth cycle of pak choi, the terminal residue levels (0.046-7.8 mg kg-1) and the relevant maximum residue limits (MRLs) of some countries, 5 d was recommended as the pre-harvest interval for the six pesticides on pak choi. Dietary risk assessment revealed that the risk quotients were 3.1%-58% for different gender and age groups in China, indicating none unacceptable public health risk for general population. The results showed that all the six pesticides degraded faster and the terminal residues were much lower under open field conditions than those under greenhouse conditions, which was mainly due to the influence of rainfall, sunlight and other environmental factors. This work was thus significant in assessing the dissipation fate and food safety risks of the six pesticides on pak choi and facilitated the establishment of maximum residue limits.

First Report of Bacterial Soft Rot Disease on Pak Choi (Brassica rapa subsp. chinensis) caused by Pectobacterium brasiliense in the United States.

Pak choi (Brassica rapa subsp. chinensis) is an important vegetable crop native to China, known for high water content and low caloric value, containing high quality of protein, carbohydrates, fiber, vitamins, minerals, and secondary plant metabolites (Acikgoz, 2016). A pak choi field (8,000 sq. ft.) on Oahu, Hawaii, was visited in May 2020. About 10% plants were infected and showed characteristic symptoms of soft rot, wet lesions, macerated infected stem and necrotic leaves (Figure1A-D); leading to the suspect of one of the most devastating bacterial pathogens within genus Pectobacterium (Boluk et al. 2020; Li et al. 2019; Arizala et al. 2020; Arizala and Arif, 2019). Four infected plants were collected from the field, and stems were surface sterilized with 0.6% sodium hypochlorite solution for 30 sec, followed by three consecutive rinses in distilled water. The stems were aseptically macerated, streaked on Crystal violet pectate medium (CVP) (Hélias et al. 2011), and incubated for 48 h at 26 ± 2°C. The peculiar morphological characteristic of pectolytic bacterial pathogen, forming pits on CVP, were observed (Meng et al. 2016) (Figure 1E). Purification of bacterial colonies were done by re-streaking of a single colony on dextrose peptone agar (DPA-without tetrazolium chloride; Norman and Alvarez 1989). DNA was isolated from bacterial cultures using the DNeasy Blood and Tissue Kit (Qiagen, Germantown, MA), respectively. Molecular identification of four strains (PL243-246) were performed by the sequencing region of the housekeeping gene dnaA (chromosomal replication initiation protein) using Pec. dnaA-F1/R1 primer set (Dobhal et al. 2020). The amplified PCR product was enzymatically cleaned using ExoSAP-ITTM (Affymetrix Inc, Santa Clara, CA), and sent for sequencing at the GENEWIZ facility (Genewiz, La Jolla, CA) using both forward and reverse primers. The dnaA gene sequences were aligned using Geneious, and manually edited to remove the errors. The consensus sequences were analyzed with the NCBI BLASTn tool and were deposited in the NCBI GenBank under the accession numbers MT899920-MT899923. The NCBI BLASTn report indicated that all the sequences shared 99-100% identity and query cover with Pectobacterium brasiliense accession numbers MN544627-29. A phylogenetic analysis, using Geneious, was performed with the dnaA sequences representing different Pectobacterium spp., all strains grouped within the clade of P. brasiliense (Figure 2; Arizala et al, 2020). A pathogenicity assay was carried out in three replications on pak choi grown in pots containing commercial pot mixture, and maintained in the controlled-greenhouse (temperature 26-30°C; relative humidity 50-58%). Three-weeks old plant stems were artificially inoculated with 100 µl bacterial suspensions of PL243 (1.3x 108 CFU/ml), PL244 (1.2x 108 CFU/ml), PL 245 (1.2x 108 CFU/ml) and PL246 (1.1x 108CFU/ml); control plants were inoculated with 100 µl of distilled water (Figure 1F). Two days after inoculation, the soft rot and wilting symptoms (Figure 1G-H), similar to the ones observed on the field, were developed for all four strains tested. Bacteria was successfully re-isolated from the inoculated plants; DNA was isolated, amplified, sequenced for dnaA region and analyzed for 100% homology with original strains, to fulfill Koch's postulates. Based on the molecular characteristics re-isolates were identical to the original strains. To the best of our knowledge, this is the first report of P. brasiliense on pak choi in the USA. Recent reports indicated that the pathogen could potentially pose a threat to cruciferous crops, therefore, highlighting a need to conduct a state-wide survey for pectinolytic bacteria, and implement better management strategies to combat the vegetable crop losses.

Blue and UV-A light wavelengths positively affected accumulation profiles of healthy compounds in pak-choi.

BACKGROUND: Recently, it become an important strategy using light to regulate plant growth and quality, especially on daily edible leafy vegetable. Pak-choi is rich in healthy functional compounds, e.g. flavonoid and glucosinolate. Many studies have focused on the plant response to increased radiation and transformed visible light quality, however, we know less about different blue and UV-A light wavelengths. Therefore, the goal of this study was to identify whether different blue and UV-A light wavelengths could improve quality in two cultivars of pak-choi and further cultivate potentially healthy functional plants. RESULTS: The different blue and UV-A light wavelength treatments significantly increased the fresh and dry weight in two cultivars of pak-choi. Compared with control, the content of soluble protein was higher after the different blue and UV-A light treatments. Similarly, the contents of total phenolics and total flavonoids increased significantly under the light treatments, and the highest content presented under T430 (supplemental blue light at 430 nm) in red-leaf pak-choi and under T400 (supplemental UV-A light at 400 nm) in green-leaf pak-choi. The total anthocyanins content and 2,2-diphenyl-1-picrylhydrazyl (DPPH) of two pak-choi cultivars improved positively with decreasing treatment wavelength, and other healthy compounds were affected to varying degrees under supplemental light treatments. CONCLUSION: The growth and healthy compound contents of pak-choi were significantly improved by supplemental blue and UV-A light, and there were wavelength- and cultivar-dependent effects. Compared with control, T430 presented the higher biomass and the contents of total phenolics, flavonoids and pigment in two pak-choi cultivars, and T380 was an efficient strategy to increase antioxidants and health-promoting compounds of red-leaf pak-choi. © 2020 Society of Chemical Industry.

Enhanced Relative Electron Transport Rate Contributes to Increased Photosynthetic Capacity in Autotetraploid Pak Choi.

Autopolyploids often show growth advantages over their diploid progenitors because of their increased photosynthetic activity; however, the underlying molecular basis of such mechanism remains elusive. In this study, we aimed to characterize autotetraploid pak choi (Brassica rapa ssp. chinensis) at the physiological, cellular and molecular levels. Autotetraploid pak choi has thicker leaves than its diploid counterparts, with relatively larger intercellular spaces and cell size and greater grana thylakoid height. Photosynthetic data showed that the relative electron transport rate (rETR) was markedly higher in autotetraploid than in diploid pak choi. Transcriptomic data revealed that the expressions of genes involved in 'photosynthesis' biological process and 'thylakoids' cellular component were mainly regulated in autotetraploids. Overall, our findings suggested that the increased rETR in the thylakoids contributed to the increased photosynthetic capacity of autotetraploid leaves. Furthermore, we found that the enhanced rETR is associated with increased BrPetC expression, which is likely altered by histone modification. The ectopic expression of BrPetC in Arabidopsis thaliana led to increased rETR and biomass, which were decreased in BrPetC-silenced pak choi. Autotetraploid pak choi also shows altered hormone levels, which was likely responsible for the increased drought resistance and the impaired powdery mildew resistance of this lineage. Our findings further our understanding on how autotetraploidy provides growth advantages to plants.

Effects of selenium combined with zinc amendment on zinc fractions and bioavailability in calcareous soil.

Selenium (Se) and zinc (Zn) are two important trace elements for human being and animals. The interaction between Se and Zn on the bioavailability of Zn in soil is still unclear. Therefore, pot experiments exposed to different dosages of zinc sulfate (ZnSO4) (0, 20, and 50 mg/kg soil) and sodium selenite (Na2SeO3) (0, 0.5, 1.0, and 2.5 mg/kg soil) were conducted to investigate the effects of selenite application on Zn bioavailability in calcareous soil and its related mechanisms. The total Zn content of different tissues (roots and shoots) of pak choi (Brassica chinensis L.) and the changes in Zn fraction distribution in soil before planting and after harvest were determined, and the mobility factor (MF) and distribution index (DI) of Zn in soils were calculated. In addition, the Pearson correlation and path analysis were conducted to clarify the relationships between Zn fractions in soil and the Zn uptake of pak choi. Results showed that Se amendment elevated soil Zn bioavailability at appropriate levels of Se and Zn. When 1.0 and 2.5 mg/kg of Se and 20 mg/kg of Zn were applied in soil, the proportion of exchangeable Zn (Ex-Zn) and Zn weakly bound to organic matter (Wbo-Zn) to the total content of Zn was significantly increased by 28.14%-82.52% compared with that of the corresponding single Zn treatment. Therefore, the Zn concentration in the shoots of pak choi was significantly increased by 27.2%-31.1%. High Zn (50 mg/kg) and Se co-amended treatments showed no significantly beneficial effect on the bioavailability of Zn. In addition, the potential available Zn content in soil (weakly bound to organic matter and carbonate bound Zn) and MF and DI values were all positively correlated with the Zn concentrations in pak choi, indicating that these indexes can be used to predict the bioavailability of Zn in soil. This study can provide a good reference for Se and Zn biofortification of plants in calcareous soil.

Cloning and Functional Analysis of BcMYB101 Gene Involved in Leaf Development in Pak Choi (Brassica rapa ssp. Chinensis).

As one of the largest transcription factor families, MYB transcription factors are widely present, and they are involved in a diverse range of physiological activities in plants, such as leaf development. GAMYB genes belong to the R2R3-MYB subfamily, which includes the MYB33/65/101 gene, and these genes are studied well in seed germination and flowering, but their roles in leaf development are poorly understood. In the current study, we isolated a GAMYB transcription factor from pak choi, BcMYB101, and analyzed its characteristics and function. The sequence structure analysis indicated that BcMYB101 has a highly conserved R2R3 DNA-binding domain in the N-terminal region and three GAMYB-specific motifs (Box1, Box2, and Box3). The expression pattern of diverse tissues revealed that BcMYB101 has a higher transcript level in the petiole, leaf, root, and floral organs. Furthermore, the expression level was significantly elevated after GA (gibberellin) treatment, suggesting that the BcMYB101 response was positively regulated by GA. Subcellular localization exhibited that BcMYB101 was only present in the nuclear region, consistent with the characterization of the transcription factor. The overexpression of BcMYB101 elucidated that BcMYB101 increased leaf number and resulted in downward-curling cauline leaves. Moreover, the virus-induced BcMYB101 silencing displayed that BcMYB101 is involved in the regulation of curly leaves. Furthermore, we discovered that BcMYB101 has two trans-activation activities and one interaction protein, BcTCH4, using a trans-activation activity assay and a yeast two-hybrid assay, respectively. In this study, we firstly isolated the BcMYB101 gene and explored its function in leaf development, thereby providing a solid foundation for further research on the regulatory mechanism of leaf shape in Brassica or other species.

Uptake and accumulation of pentachloronitrobenzene in pak choi and the human health risk.

Nowadays, nanocarbon is widely employed to enwrap into fertilizers. However, the influence of nanocarbon on the transportation of contaminants from soil to plants and its mechanism remain unclear. In this study, pentachloronitrobenzene (PCNB), a typical organochlorine fungicide utilized all over the world, was chosen as the target contaminant to investigate the influence of nanocarbon on its transportation in soil-pak choi system. The maximum PCNB concentration in the root and leaf reached to 112 and 86 ng/g, respectively, demonstrating that PCNB would be absorbed by pak choi. The ratio of PCNB between leaf and root indicated that nanocarbon promoted root of pak choi to absorb PCNB. The transportation of PCNB inside plant was inhibited when pak choi was planted in soil containing higher concentration of nanocarbon. Human risk assessment showed that people consuming the pak choi in this study would not experience risk. However, in vitro toxicity test indicated that PCNB could directly impair intestinal epithelial cells (Caco-2 cells) and thus pose a potential risk to human intestine.

BcMAF2 activates BcTEM1 and represses flowering in Pak-choi (Brassica rapa ssp. chinensis).

KEY MESSAGE: BcMAF2 plays a key role in flowering regulation by controlling BcTEM1, BcSOC1 and BCSPL15 in Pak-choi. Flowering is a key event in the life cycle of plants. Flowering time shows an extensive variation from different Pak-choi (Brassica rapa ssp. chinensis) cultivars. However, the regulation mechanism of flowering in Pak-choi remains rarely known. In this study, a systematic identification and functional analysis of a Pak-choi MADS Affecting Flowering (MAF) gene, BcMAF2, was carried out. BcMAF2 encoded a protein containing a conserved MADS-box domain, which was localized in the nucleus. QPCR analysis indicated that the expression of BcMAF2 was higher in the leaves and flowers. Overexpression of BcMAF2 in Arabidopsis showed that BcMAF2 repressed flowering, which was further confirmed by silencing endogenous BcMAF2 in Pak-choi. In addition, Tempranillo 1 (TEM1) expression was up-regulated and MAF2 expression was down-regulated in the BcMAF2-overexpressing Arabidopsis. The expression of BcMAF2 and BcTEM1 was down-regulated in BcMAF2-silencing Pak-choi plants. The yeast one-hybrid, dual luciferase and qPCR results revealed that BcMAF2 protein could directly bind to BcTEM1 promoter and activate its expression, which was not reported in Arabidopsis. Meanwhile, a self-inhibition was found in BcMAF2. Taken together, this work suggested that BcMAF2 could repress flowering by directly activating BcTEM1.

1-Methoxy-3-indolylmethyl DNA adducts in six tissues, and blood protein adducts, in mice under pak choi diet: time course and persistence.

We previously showed that purified 1-methoxy-3-indolylmethyl (1-MIM) glucosinolate, a secondary plant metabolite in Brassica species, is mutagenic in various in vitro systems and forms DNA and protein adducts in mouse models. In the present study, we administered 1-MIM glucosinolate in a natural matrix to mice, by feeding a diet containing pak choi powder and extract. Groups of animals were killed after 1, 2, 4 and 8 days of pak choi diet, directly or, in the case of the 8-day treatment, after 0, 8 and 16 days of recovery with pak choi-free diet. DNA adducts [N2-(1-MIM)-dG, N6-(1-MIM)-dA] in six tissues, as well as protein adducts [τN-(1-MIM)-His] in serum albumin (SA) and hemoglobin (Hb) were determined using UPLC-MS/MS with isotopically labeled internal standards. None of the samples from the 12 control animals under standard diet contained any 1-MIM adducts. All groups receiving pak choi diet showed DNA adducts in all six tissues (exception: lung of mice treated for a single day) as well as SA and Hb adducts. During the feeding period, all adduct levels continuously increased until day 8 (in the jejunum until day 4). During the 14-day recovery period, N2-(1-MIM)-dG in liver, kidney, lung, jejunum, cecum and colon decreased to 52, 41, 59, 11, 7 and 2%, respectively, of the peak level. The time course of N6-(1-MIM)-dA was similar. Immunohistochemical analyses indicated that cell turnover is a major mechanism of DNA adduct elimination in the intestine. In the same recovery period, protein adducts decreased more rapidly in SA than in Hb, to 0.7 and 37%, respectively, of the peak level, consistent with the differential turnover of these proteins. In conclusion, the pak choi diet lead to the formation of high levels of adducts in mice. Cell and protein turnover was a major mechanism of adduct elimination, at least in gut and blood.