Transcriptomic analysis of succulent stem development of Chinese kale (Brassica oleracea var. alboglabra Bailey) and its synthetic allotetraploid via RNA sequencing.

Chinese kale (Brassica oleracea var. alboglabra Bailey, CC) is a succulent stem vegetable in the Brassica family. Its allotetraploid (AACC) vegetable germplasm, which was synthesized via distant hybridization with the colloquially named 'yellow turnip' (B. rapa L. ssp. rapifera Matzg., AA), has a swelling stem similar to CC. To address the molecular mechanism of stem development for CC and AACC, RNA sequencing (RNA-seq) was used to investigate transcriptional regulation of their stem development at three key stages including 28 days, 42 days and the bolting stage (BS) after sowing. As a result, 32,642, 32,665, 33,816, 32,147, 32,293 and 32,275 genes were identified in six corresponding cDNA libraries. Among them, 25,459 genes were co-expressed, while 7,183, 7,206, 8,357, 6,688, 6,834 and 6,814 genes were specifically expressed. Additionally, a total of 29,222 differentially expressed genes (DEGs) were found for functional enrichment as well as many genes involved in plant hormones including gibberellin (GA), abscisic acid (ABA), cytokinin (CTK) and auxin (AUX). Based on gene expression consistency between CC and AACC, the gene families including DELLA, GID, PYR/PYL, PP2C, A-ARR and AUX/IAA might be related to stem development. Among these, eight genes including Bo00834s040, Bo5g093140, Bo6g086770, Bo9g070200, Bo7g116570, Bo3g054410, Bo7g093470 and Bo5g136600 may play important roles in stem development based on their remarkable expression levels as confirmed by qRT-PCR. These findings provide a new theoretical basis for understanding the molecular mechanism of stem development in Brassica vegetable stem breeding.

Leaf and root glucosinolate profiles of Chinese cabbage (Brassica rapa ssp. pekinensis) as a systemic response to methyl jasmonate and salicylic acid elicitation.

Glucosinolates (GSs) are an important group of defensive phytochemicals mainly found in Brassicaceae. Plant hormones jasmonic acid (JA) and salicylic acid (SA) are major regulators of plant response to pathogen attack. However, there is little information about the interactive effect of both elicitors on inducing GS biosynthesis in Chinese cabbage (Brassica rapa ssp. pekinensis). In this study, we applied different concentrations of methyl jasmonate (MeJA) and/or SA onto the leaf and root of Chinese cabbage to investigate the time-course interactive profiles of GSs. Regardless of the site of the elicitation and the concentrations of the elicitors, the roots accumulated much more GSs and were more sensitive and more rapidly responsive to the elicitors than leaves. Irrespective of the elicitation site, MeJA had a greater inducing and longer lasting effect on GS accumulation than SA. All three components of indole GS (IGS) were detected along with aliphatic and aromatic GSs. However, IGS was a major component of total GSs that accumulated rapidly in both root and leaf tissues in response to MeJA and SA elicitation. Neoglucobrassicin (neoGBC) did not respond to SA but to MeJA in leaf tissue, while it responded to both SA and MeJA in root tissue. Conversion of glucobrassicin (GBC) to neoGBC occurred at a steady rate over 3 d of elicitation. Increased accumulation of 4-methoxy glucobrassicin (4-MGBC) occurred only in the root irrespective of the type of elicitors and the site of elicitation. Thus, accumulation of IGS is a major metabolic hallmark of SA- and MeJA-mediated systemic response systems. SA exerted an antagonistic effect on the MeJA-induced root GSs irrespective of the site of elicitation. However, SA showed synergistic and antagonistic effects on the MeJA-induced leaf GSs when roots and leaves are elicitated for 3 d, respectively.

Genome-wide identification of glucosinolate synthesis genes in Brassica rapa.

Glucosinolates play important roles in plant defense against herbivores and microbes, as well as in human nutrition. Some glucosinolate-derived isothiocyanate and nitrile compounds have been clinically proven for their anticarcinogenic activity. To better understand glucosinolate biosynthesis in Brassica rapa, we conducted a comparative genomics study with Arabidopsis thaliana and identified total 56 putative biosynthetic and regulator genes. This established a high colinearity in the glucosinolate biosynthesis pathway between Arabidopsis and B. rapa. Glucosinolate genes in B. rapa share 72-94% nucleotide sequence identity with the Arabidopsis orthologs and exist in different copy numbers. The exon/intron split pattern of B. rapa is almost identical to that of Arabidopsis, although inversion, insertion, deletion and intron size variations commonly occur. Four genes appear to be nonfunctional as a result of the presence of a frame shift mutation and retrotransposon insertion. At least 12 paralogs of desulfoglucosinolate sulfotransferase were found in B. rapa, whereas only three were found in Arabidopsis. The expression of those paralogs was not tissue-specific but varied greatly depending on B. rapa tissue types. Expression was also developmentally regulated in some paralogs but not in other paralogs. Most of the regulator genes are present as triple copies. Accordingly, glucosinolate synthesis and regulation in B. rapa appears to be more complex than that of Arabidopsis. With the isolation and further characterization of the endogenous genes, health-beneficial vegetables or desirable animal feed crops could be developed by metabolically engineering the glucosinolate pathway.

Metabolic engineering of aliphatic glucosinolates in Chinese cabbage plants expressing Arabidopsis MAM1, CYP79F1, and CYP83A1.

Three Arabidopsis cDNAs, MAM1, CYP79F1, and CYP83A1, required for aliphatic glucosinolate biosynthesis were introduced into Chinese cabbage by Agrobacterium tumefaciens-mediated transformation. The transgenic lines overexpressing MAM1 or CYP83A1 showed wild-type phenotypes. However, all the lines overexpressing CYP79F1 displayed phenotypes different from wild type with respect to the stem thickness as well as leaf width and shape. Glucosinolate contents of the transgenic plants were compared with those of wild type. In the MAM1 line M1-1, accumulation of aliphatic glucosinolates gluconapin and glucobrassicanapin significantly increased. In the CYP83A1 line A1-1, all the aliphatic glucosinolate levels were increased, and the levels of gluconapin and glucobrassicanapin were elevated by 4.5 and 2 fold, respectively. The three CYP79F1 transgenic lines exhibited dissimilar glucosinolate profiles. The F1-1 line accumulated higher levels of gluconapoleiferin, glucobrassicin, and 4-methoxy glucobrassicin. However, F1-2 and F1-3 lines demonstrated a decrease in the levels of gluconapin and glucobrassicanapin and an increased level of 4-hydroxy glucobrassicin.

Metabolic engineering of indole glucosinolates in Chinese cabbage plants by expression of Arabidopsis CYP79B2, CYP79B3, and CYP83B1.

Indole glucosinolates (IG) play important roles in plant defense, plant-insect interactions, and stress responses in plants. In an attempt to metabolically engineer the IG pathway flux in Chinese cabbage, three important Arabidopsis cDNAs, CYP79B2, CYP79B3, and CYP83B1, were introduced into Chinese cabbage by Agrobacterium-mediated transformation. Overexpression of CYP79B3 or CYP83B1 did not affect IG accumulation levels, and overexpression of CYP79B2 or CYP79B3 prevented the transformed callus from being regenerated, displaying the phenotype of indole-3-acetic acid (IAA) overproduction. However, when CYP83B1 was overexpressed together with CYP79B2 and/or CYP79B3, the transformed calli were regenerated into whole plants that accumulated higher levels of glucobrassicin, 4-hydroxy glucobrassicin, and 4-methoxy glu-cobrassicin than wild-type controls. This result suggests that the flux in Chinese cabbage is predominantly channeled into IAA biosynthesis so that coordinate expression of the two consecutive enzymes is needed to divert the flux into IG biosynthesis. With regard to IG accumulation, overexpression of all three cDNAs was no better than overexpression of the two cDNAs. The content of neoglucobrassicin remained unchanged in all transgenic plants. Although glucobrassicin was most directly affected by overexpression of the transgenes, elevated levels of the parent IG, glucobrassicin, were not always accompanied by increases in 4-hydroxy and 4-methoxy glucobrassicin. However, one transgenic line producing about 8-fold increased glucobrassicin also accumulated at least 2.5 fold more 4-hydroxy and 4-methoxy glucobrassicin. This implies that a large glucobrassicin pool exceeding some threshold level drives the flux into the side chain modification pathway. Aliphatic glucosinolate content was not affected in any of the transgenic plants.