Search

Plant phosphoprotein phosphatases are ubiquitous and multifarious enzymes that respond to developmental requirements and stress signals through reversible dephosphorylation of target proteins. In this study, we investigated the hitherto unknown functions of Brassica rapa protein phosphatase 5.2 ( BrPP5.2 ) by transgenic overexpression of B. rapa lines. The overexpression of BrPP5.2 in transgenic lines conferred heat shock tolerance in 65-89% of the young transgenic seedlings exposed to 46 °C for 25 min. The examination of purified recombinant BrPP5.2 at different molar ratios efficiently prevented the thermal aggregation of malate dehydrogenase at 42 °C, thus suggesting that BrPP5.2 has inherent chaperone activities. The transcriptomic dynamics of transgenic lines, as determined using RNA-seq, revealed that 997 and 1206 (FDR < 0.05, logFC ≥ 2) genes were up- and down-regulated, as compared to non-transgenic controls. Statistical enrichment analyses revealed abiotic stress response genes, including heat stress response (HSR), showed reduced expression in transgenic lines under optimal growth conditions. However, most of the HSR DEGs were upregulated under high temperature stress (37 °C/1 h) conditions. In addition, the glucosinolate biosynthesis gene expression and total glucosinolate content increased in the transgenic lines. These findings provide a new avenue related to BrPP5.2 downstream genes and their crucial metabolic and heat stress responses in plants.

Glucoraphasatin (GRH) is a specific aliphatic glucosinolate (GSL) that is only abundant in radish ( Raphanus sativus L.). The gene expression regulating GRH biosynthesis in radish is still poorly understood. We employed a total of 59 radish accessions to analyze GSL profiles and showed that GRH was specific and predominant among the aliphatic GSLs in radish roots. We selected five accessions roots with high, moderate and low GSL biosynthesis, respectively, to conduct a comparative transcriptome analysis and the qRT-PCR of the biosynthesis genes for aliphatic GSLs. In this study, among all the accessions tested, roots with the accession RA157-74 had a high GRH content and showed a significant expression of the aliphatic GSL biosynthesis genes. We defined the genes involved in the GRH biosynthesis process and found that they were regulated by a transcription factor ( RSG00789 ) at the MYB29 locus in radish roots. We found 13 aliphatic GSL biosynthesis genes regulated by the RSG00789 gene in the GRH biosynthesis pathway.

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. Here, we present a shot-gun differential proteomic analysis on roots of two Arabidopsis pldα1 mutants compared to the wild type. Interestingly, PLDα1 deficiency leads to altered abundances of proteins involved in diverse processes related to membrane transport including endocytosis and endoplasmic reticulum-Golgi transport. PLDα1 may be involved in the stability of attachment sites of endoplasmic reticulum to the plasma membrane as suggested by increased abundance of synaptotagmin 1, which was validated by immunoblotting and whole-mount immunolabelling analyses. Moreover, we noticed a robust abundance alterations of proteins involved in mitochondrial import and electron transport chain. Notably, the abundances of numerous proteins implicated in glucosinolate biosynthesis were also affected in pldα1 mutants. Our results suggest a broader biological involvement of PLDα1 than anticipated thus far, especially in the processes such as endomembrane transport, mitochondrial protein import and protein quality control, as well as glucosinolate biosynthesis.

Sclerotinia stem rot (white mold), caused by the fungus Sclerotinia sclerotiorum , is a serious disease of Brassica crops worldwide. Despite considerable progress in investigating plant defense mechanisms against this pathogen, which have revealed the involvement of glucosinolates, the host⁻pathogen interaction between cabbage ( Brassica oleracea ) and S. sclerotiorum has not been fully explored. Here, we investigated glucosinolate profiles and the expression of glucosinolate biosynthesis genes in white-mold-resistant (R) and -susceptible (S) lines of cabbage after infection with S. sclerotiorum . The simultaneous rise in the levels of the aliphatic glucosinate glucoiberverin (GIV) and the indolic glucosinate glucobrassicin (GBS) was linked to white mold resistance in cabbage. Principal component analysis showed close association between fungal treatment and cabbage GIV and GBS contents. The correlation analysis showed significant positive associations between GIV content and expression of the glucosinolate biosynthesis genes ST5b-Bol026202 and ST5c-Bol030757 , and between GBS content and the expression of the glucosinolate biosynthesis genes ST5a-Bol026200 and ST5a-Bol039395 . Our results revealed that S. sclerotiorum infection of cabbage induces the expression of glucosinolate biosynthesis genes, altering the content of individual glucosinolates. This relationship between the expression of glucosinolate biosynthesis genes and accumulation of the corresponding glucosinolates and resistance to white mold extends the molecular understanding of glucosinolate-negotiated defense against S. sclerotiorum in cabbage.

Ringspot, caused by the fungus Mycosphaerella brassicicola , is a serious disease of Brassica crops worldwide. Despite noteworthy progress to reveal the role of glucosinolates in pathogen defense, the host⁻pathogen interaction between cabbage ( Brassica oleracea ) and M. brassicicola has not been fully explored. Here, we investigated the glucosinolate profiles and expression of glucosinolate biosynthesis genes in the ringspot-resistant (R) and susceptible (S) lines of cabbage after infection with M. brassicicola . The concomitant rise of aliphatic glucoiberverin (GIV) and indolic glucobrassicin (GBS) and methoxyglucobrassicin (MGBS) was linked with ringspot resistance in cabbage. Pearson's correlation and principle component analysis showed a significant positive association between GIV contents and the expression of the glucosinolate biosynthesis gene ST5b-Bol026202 and between GBS contents and the expression of the glucosinolate biosynthesis gene MYB34-Bol017062 . Our results confirmed that M. brassicicola infection induces the expression of glucosinolate biosynthesis genes in cabbage, which alters the content of individual glucosinolates. This link between the expression of glucosinolate biosynthesis genes and the accumulation of their respective glucosinolates with the resistance to ringspot extends our molecular sense of glucosinolate-negotiated defense against M. brassicicola in cabbage.

Broccoli sprouts contain health-promoting phytochemicals that can be enhanced by applying ultraviolet light (UV) or phytohormones. The separate and combined effects of methyl jasmonate (MJ), UVA, or UVB lights on glucosinolate, phenolic, carotenoid, and chlorophyll profiles were assessed in broccoli sprouts. Seven-day-old broccoli sprouts were exposed to UVA (9.47 W/m²) or UVB (7.16 W/m²) radiation for 120 min alone or in combination with a 25 µM MJ solution, also applied to sprouts without UV supplementation. UVA + MJ and UVB + MJ treatments increased the total glucosinolate content by ~154% and ~148%, respectively. MJ induced the biosynthesis of indole glucosinolates, especially neoglucobrassicin (~538%), showing a synergistic effect with UVA stress. UVB increased the content of aliphatic and indole glucosinolates, such as glucoraphanin (~78%) and 4-methoxy-glucobrassicin (~177%). UVA increased several phenolics such as gallic acid (~57%) and a kaempferol glucoside (~25.4%). MJ treatment decreased most phenolic levels but greatly induced accumulation of 5-sinapoylquinic acid (~239%). MJ treatments also reduced carotenoid and chlorophyll content, while UVA increased lutein (~23%), chlorophyll b (~31%), neoxanthin (~34%), and chlorophyll a (~67%). Results indicated that UV- and/or MJ-treated broccoli sprouts redirect the carbon flux to the biosynthesis of specific glucosinolates, phenolics, carotenoids, and chlorophylls depending on the type of stress applied.

Pak choi plants (Brassica rapa ssp. chinensis) were treated with different signaling molecules methyl jasmonate, jasmonic acid, linolenic acid, and methyl salicylate and were analyzed for specific changes in their glucosinolate profile. Glucosinolate levels were quantified using HPLC-DAD-UV, with focus on induction of indole glucosinolates and special emphasis on 1-methoxy-indol-3-ylmethyl glucosinolate. Furthermore, the effects of the different signaling molecules on indole glucosinolate accumulation were analyzed on the level of gene expression using semi-quantitative realtime RT-PCR of selected genes. The treatments with signaling molecules were performed on sprouts and mature leaves to determine ontogenetic differences in glucosinolate accumulation and related gene expression. The highest increase of indole glucosinolate levels, with considerable enhancement of the 1-methoxy-indol-3-ylmethyl glucosinolate content, was achieved with treatments of sprouts and mature leaves with methyl jasmonate and jasmonic acid. This increase was accompanied by increased expression of genes putatively involved in the indole glucosinolate biosynthetic pathway. The high levels of indole glucosinolates enabled the plant to preferentially produce the respective breakdown products after tissue damage. Thus, pak choi plants treated with methyl jasmonate or jasmonic acid, are a valuable tool to analyze the specific protection functions of 1-methoxy-indole-3-carbinole in the plants defense strategy in the future.