Widely-targeted metabolomics and transcriptomics identify metabolites associated with flowering regulation of Choy Sum.

Choy Sum, a stalk vegetable highly valued in East and Southeast Asia, is characterized by its rich flavor and nutritional profile. Metabolite accumulation is a key factor in Choy Sum stalk development; however, no research has focused on metabolic changes during the development of Choy Sum, especially in shoot tip metabolites, and their effects on growth and flowering. Therefore, in the present study, we used a widely targeted metabolomic approach to analyze metabolites in Choy Sum stalks at the seedling (S1), bolting (S3), and flowering (S5) stages. In total, we identified 493 metabolites in 31 chemical categories across all three developmental stages. We found that the levels of most carbohydrates and amino acids increased during stalk development and peaked at S5. Moreover, the accumulation of amino acids and their metabolites was closely related to G6P, whereas the expression of flowering genes was closely related to the content of T6P, which may promote flowering by upregulating the expressions of BcSOC1, BcAP1, and BcSPL5. The results of this study contribute to our understanding of the relationship between the accumulation of stem tip substances during development and flowering and of the regulatory mechanisms of stalk development in Choy Sum and other related species.

Comparative transcriptomic analysis provides key genetic resources in clove basil (Ocimum gratissimum) under cadmium stress.

Planting aromatic plant might be a promising strategy for safely utilizing heavy metal (HM)-contaminated soils, as HMs in essential oil could be completely excluded using some special technologies with ease. Clove basil (Ocimum gratissimum L.) is an important aromatic plant used in essential oil production. Improving cadmium (Cd) tolerance in clove basil can increase its production and improve the utilization efficiency of Cd-contaminated soils. However, the lack of genomic information on clove basil greatly restricts molecular studies and applications in phytoremediation. In this study, we demonstrated that high levels of Cd treatments (0.8, 1.6 and 6.5 mg/L) significantly impacted the growth and physiological attributes of clove basil. Cd contents in clove basil tissues increased with treatment concentrations. To identify Cd stress-responsive genes, we conducted a comparative transcriptomic analysis using seedlings cultured in the Hoagland's solution without Cd ion (control) or containing 1.6 mg/L CdCl2 (a moderate concentration of Cd stress for clove basil seedlings). A total of 104.38 Gb clean data with high-quality were generated in clove basil under Cd stress through Illumina sequencing. More than 1,800 differential expressed genes (DEGs) were identified after Cd treatment. The reliability and reproducibility of the transcriptomic data were validated through qRT-PCR analysis and Sanger sequencing. KEGG classification analysis identified the "MAPK signaling pathway," "plant hormone signal transduction" and "plant-pathogen interaction" as the top three pathways. DEGs were divided into five clusters based on their expression patterns during Cd stress. The functional annotation of DEGs indicated that downregulated DEGs were mainly involved in the "photosynthesis system," whereas upregulated DEGs were significantly assigned to the "MAPK signaling pathway" and "plant-pathogen interaction pathway." Furthermore, we identified a total of 78 transcription factors (TFs), including members of bHLH, WRKY, AP2/ERF, and MYB family. The expression of six bHLH genes, one WRKY and one ERF genes were significantly induced by Cd stress, suggesting that these TFs might play essential roles in regulating Cd stress responses. Overall, our study provides key genetic resources and new insights into Cd adaption mechanisms in clove basil.

Low-level cadmium exposure induced hormesis in peppermint young plant by constantly activating antioxidant activity based on physiological and transcriptomic analyses.

As one of the most toxic environmental pollutants, cadmium (Cd) has lastingly been considered to have negative influences on plant growth and productivity. Recently, increasing studies have shown that low level of Cd exposure could induce hormetic effect which benefits to plants. However, the underlying mechanisms of Cd-triggered hormesis are poorly understood. In this study, we found that Cd stress treatment showed a hormetic effect on peppermint and Cd treatment with 1.6 mg L-1 concertation manifested best stimulative effects. To explore the hormesis mechanisms of Cd treatment, comparative transcriptome analysis of peppermint young plants under low (1.6 mg L-1) and high (6.5 mg L-1) level of Cd exposure at 0 h, 24 h and 72 h were conducted. Twelve of differentially expressed genes (DEGs) were selected for qRT-PCR validation, and the expression results confirmed the credibility of transcriptome data. KEGG analysis of DEGs showed that the phenylpropanoid biosynthesis and photosynthesis were important under both low and high level of Cd treatments. Interestingly, GO and KEGG analysis of 99 DEGs specifically induced by low level of Cd treatment at 72 h indicated that these DEGs were mainly involved in the pathway of phenylpropanoid biosynthesis and their functions were associated with antioxidant activity. The expression pattern of those genes in the phenylpropanoid biosynthesis pathway and encoding antioxidant enzymes during 72 h of Cd exposure showed that low level of Cd treatment induced a continuation in the upward trend but high level of Cd treatment caused an inverted V-shape. The changes of physiological parameters during Cd exposure were highly consistent with gene expression pattern. These results strongly demonstrate that low level of Cd exposure constantly enhanced antioxidant activity of peppermint to avoid oxidative damages caused by Cd ion, while high level of Cd stress just induced a temporary increase in antioxidant activity which was insufficient to cope with lasting Cd toxicity. Overall, the results presented in this study shed a light on the underlying mechanisms of the Cd-mediated hormesis in plant. Moreover, our study provided a safe method for the efficient utilization of mild Cd-contaminated soil as peppermint is an important cash plant.

Unraveling the Mechanism of Purple Leaf Formation in Brassica napus by Integrated Metabolome and Transcriptome Analyses.

Brassica napus as both oilseed and vegetable, is widely cultivated in China. The purple leaf of B. napus is rich in anthocyanins and can provide valuable nutrients. Although several high-anthocyanin cultivars have been reported, the molecular mechanism underlying anthocyanin biosynthesis in B. napus remains lesser-known. Therefore, in this study, we conducted integrative metabolome and transcriptome analyses in three B. napus cultivars with different leaf colors. Overall, 39 flavonoids were identified (including 35 anthocyanins), and 22 anthocyanins were differentially accumulated in the leaves, contributing to the different leaf colors. Cyanidin-3,5,3'-O-triglucoside was confirmed as the main contributor of the purple leaf phenotype. Meanwhile, other anthocyanins may play important roles in deepening the color of B. napus leaves. A total of 5,069 differentially expressed genes (DEGs) and 32 overlapping DEGs were identified by RNA-sequencing; hence, the correlation between anthocyanin content and DEG expression levels was explored. Two structural genes (DFR and ANS), three GSTs (homologous to TT19), and 68 differentially expressed transcription factors (TFs), especially MYB-related TFs and WRKY44, were identified in three B. napus varieties characterized by different leaf color, thereby indicating that these genes may contribute to anthocyanin biosynthesis, transport, or accumulation in B. napus leaves. The findings of study provide important insights that may contribute to gaining a better understanding of the transcriptional regulation of anthocyanin metabolism in B. napus.

Identification of DELLA Genes and Key Stage for GA Sensitivity in Bolting and Flowering of Flowering Chinese Cabbage.

Flowering Chinese cabbage (Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee) is an important and extensively cultivated vegetable in south China, and its stalk development is mainly regulated by gibberellin (GA). DELLA proteins negatively regulate GA signal transduction and may play an important role in determining bolting and flowering. Nevertheless, no systematic study of the DELLA gene family has been undertaken in flowering Chinese cabbage. In the present study, we found that the two-true-leaf spraying of gibberellin A3 (GA3) did not promote bolting but did promote flowering, whereas the three-true-leaf spraying of GA3 promoted both bolting and flowering. In addition, we identified five DELLA genes in flowering Chinese cabbage. All five proteins contained DELLA, VHYNP, VHIID, and SAW conserved domains. Protein-protein interaction results showed that in the presence of GA3, all five DELLA proteins interacted with BcGID1b (GA-INSENSITIVE DWARF 1b) but not with BcGID1a (GA-INSENSITIVE DWARF 1a) or BcGID1c (GA-INSENSITIVE DWARF 1c). Their expression analysis showed that the DELLA genes exhibited tissue-specific expression, and their reversible expression profiles responded to exogenous GA3 depending on the treatment stage. We also found that the DELLA genes showed distinct expression patterns in the two varieties of flowering Chinese cabbage. BcRGL1 may play a major role in the early bud differentiation process of different varieties, affecting bolting and flowering. Taken together, these results provide a theoretical basis for further dissecting the DELLA regulatory mechanism in the bolting and flowering of flowering Chinese cabbage.

Appropriate NH4 +/NO3 - Ratio Triggers Plant Growth and Nutrient Uptake of Flowering Chinese Cabbage by Optimizing the pH Value of Nutrient Solution.

Compared with sole nitrogen (N), the nutrition mixture of ammonium (NH4 +) and nitrate (NO3 -) is known to better improve crop yield and quality. However, the mechanism underlying this improvement remains unclear. In the present study, we analyzed the changes in nutrient solution composition, content of different N forms in plant tissues and exudates, and expression of plasma membrane (PM) H+-ATPase genes (HAs) under different NH4 +/NO3 - ratios (0/100, 10/90, 25/75, 50/50 as control, T1, T2, and T3) in flowering Chinese cabbage. We observed that compared with the control, T1 and T2 increased the economical yield of flowering Chinese cabbage by 1.26- and 1.54-fold, respectively, whereas T3 significantly reduced plant yield. Compared with the control, T1-T3 significantly reduced the NO3 - content and increased the NH4 +, amino acid, and soluble protein contents of flowering Chinese cabbage to varying extents. T2 significantly increased the N use efficiency (NUE), whereas T3 significantly decreased it to only being 70.25% of that of the control. Owing to the difference in N absorption and utilization among seedlings, the pH value of the nutrient solution differed under different NH4 +/NO3 - ratios. At harvest, the pH value of T2 was 5.8; in the control and T1, it was approximately 8.0, and in T3 it was only 3.6. We speculated that appropriate NH4 +/NO3 - ratios may improve N absorption and assimilation and thus promote the growth of flowering Chinese cabbage, owing to the suitable pH value. On the contrary, addition of excessive NH4 + may induce rhizosphere acidification and ammonia toxicity, causing plant growth inhibition. We further analyzed the transcription of PM H+-ATPase genes (HAs). HA1 and HA7 transcription in roots was significantly down-regulated by the addition of the mixture of NH4 + and NO3 -, whereas the transcription of HA2, HA9 in roots and HA7, HA8, and HA10 in leaves was sharply up-regulated by the addition of the mixture; the transcription of HA3 was mainly enhanced by the highest ratio of NH4 +/NO3 -. Our results provide valuable information about the effects of treatments with different NH4 +/NO3 - ratios on plant growth and N uptake and utilization.

Crosstalk between auxin and gibberellin during stalk elongation in flowering Chinese cabbage.

Plant growth and development are tightly regulated by phytohormones. However, little is known about the interaction between auxin and gibberellin acid (GA) during flower stalk elongation and how it is directly related to organ formation. Therefore, the effects of indole acetic acid (IAA) and GA3 treatments and their interaction on flower stalk elongation in flowering Chinese cabbage were investigated. The growth of flowering Chinese cabbage is regulated by IAA and GA3, and the opposite results were observed after treatments with uniconazole (GA synthesis inhibitor) and N-1-naphthylphthalamic acid (NPA) (auxin transport inhibitor). Anatomical analysis of the pith region in stalks revealed that IAA promoted expansion via signal transduction and transport pathways. GA3 regulated the elongation of flower stalks by controlling GA synthesis and partially controlling the IAA signaling pathway. GA3 also had a stronger effect on stalk elongation than IAA. The results of qRT-PCR and histological analysis revealed that GA3 and IAA induced the expansion of cell walls by activating the expression of genes encoding cell wall structural proteins such as Expansin (EXP). These findings provide new insights into the mechanism of stalk formation regulated by the combination of IAA and GA3.

Ammonium Transporter (BcAMT1.2) Mediates the Interaction of Ammonium and Nitrate in Brassica campestris.

The provision of ammonium (NH4 +) and nitrate (NO3 -) mixture increases the total nitrogen (N) than the supply of sole NH4 + or NO3 - with the same concentration of total N; thus, the mixture contributes to better growth in Brassica campestris. However, the underlying mechanisms remain unknown. In this study, we analyzed NH4 + and NO3 - fluxes using a scanning ion-selective electrode technique to detect under different N forms and levels in B. campestris roots. We observed that the total N influxes with NH4 + and NO3 - mixture were 1.25- and 3.53-fold higher than those with either sole NH4 + or NO3 -. Furthermore, NH4 + and NO3 - might interact with each other under coexistence. NO3 - had a positive effect on net NH4 + influx, whereas NH4 + had a negative influence on net NO3 - influx. The ammonium transporter (AMT) played a key role in NH4 + absorption and transport. Based on expression analysis, BcAMT1.2 differed from other BcAMT1s in being upregulated by NH4 + or NO3 -. According to sequence analysis and functional complementation in yeast mutant 31019b, AMT1.2 from B. campestris may be a functional AMT. According to the expression pattern of BcAMT1.2, ß-glucuronidase activity, and the cellular location of its promoter, BcAMT1.2 may be responsible for NH4 + transport. Following the overexpression of BcAMT1.2 in Arabidopsis, BcAMT1.2-overexpressing lines grew better than wildtype lines at low NH4 + concentration. In the mixture of NH4 + and NO3 -, NH4 + influxes and NO3 - effluxes were induced in BcAMT1.2-overexpressing lines. Furthermore, transcripts of N assimilation genes (AtGLN1.2, AtGLN2, and AtGLT1) were significantly upregulated, in particular, AtGLN1.2 and AtGLT1 were increased by 2.85-8.88 times in roots, and AtGLN1.2 and AtGLN2 were increased by 2.67-4.61 times in leaves. Collectively, these results indicated that BcAMT1.2 may mediate in NH4 + fluxes under the coexistence of NH4 + and NO3 - in B. campestris.

Identification and characterization of two ammonium transporter genes in flowering Chinese cabbage (Brassica campestris).

Ammonium transporters (AMTs), which include AMT1 and AMT2 subfamilies, have been identified and partially characterized in many plants. In this study, two AMT2-type genes from Brassica campestris, namely BcAMT2 and BcAMT2like, were identified and characterized. BcAMT2 and BcAMT2like are 2666 bp and 2952 bp, encode proteins of 490 and 489 amino acids, respectively, and contain five exons and four introns. Transient expression of these proteins labelled with green fluorescence protein in onion epidermal cells indicated that both are located on the plasma membrane. When expressing BcAMT2 or BcAMT2like, the mutant yeast strain 31019b could grow on medium containing 2 mM ammonium as the only nitrogen source when expressing BcAMT2 or BcAMT2like, indicating that both are functional AMT genes. Quantitative PCR results showed that BcAMT2 and BcAMT2like were expressed in all tissues, but they displayed different expression patterns in the reproductive stages. BcAMT2s transcript levels in leaves were positively correlated with ammonium concentration and external pH. Moreover, the expression BcAMT2s responded to diurnal change. Furthermore, the uncharged form of ammonium, i.e., ammonia, might also be transported by BcAMT2s. These results provide new insights into the molecular mechanisms underlying ammonium absorption and transportation by the AMT2 subfamily in B. campestris.