Downregulation of Brassica napus MYB69 (BnMYB69) increases biomass growth and disease susceptibility via remodeling phytohormone, chlorophyll, shikimate and lignin levels

MYB transcription factors are major actors regulating plant development and adaptability. Brassica napus is a staple oil crop and is hampered by lodging and diseases. Here, four B. napus MYB69 (BnMYB69s) genes were cloned and functionally characterized. They were dominantly expressed in stems during lignification. BnMYB69 RNA interference (BnMYB69i) plants showed considerable changes in morphology, anatomy, metabolism and gene expression. Stem diameter, leaves, roots and total biomass were distinctly larger, but plant height was significantly reduced. Contents of lignin, cellulose and protopectin in stems were significantly reduced, accompanied with decrease in bending resistance and Sclerotinia sclerotiorum resistance. Anatomical detection observed perturbation in vascular and fiber differentiation in stems, but promotion in parenchyma growth, accompanied with changes in cell size and cell number. In shoots, contents of IAA, shikimates and proanthocyanidin were reduced, while contents of ABA, BL and leaf chlorophyll were increased. qRT-PCR revealed changes in multiple pathways of primary and secondary metabolisms. IAA treatment could recover many phenotypes and metabolisms of BnMYB69i plants. However, roots showed trends opposite to shoots in most cases, and BnMYB69i phenotypes were light-sensitive. Conclusively, BnMYB69s might be light-regulated positive regulators of shikimates-related metabolisms, and exert profound influences on various internal and external plant traits

Gene silencing of BnTT10 family genes causes retarded pigmentation and lignin reduction in the seed coat of Brassica napus.

Yellow-seed (i.e., yellow seed coat) is one of the most important agronomic traits of Brassica plants, which is correlated with seed oil and meal qualities. Previous studies on the Brassicaceae, including Arabidopsis and Brassica species, proposed that the seed-color trait is correlative to flavonoid and lignin biosynthesis, at the molecular level. In Arabidopsis thaliana, the oxidative polymerization of flavonoid and biosynthesis of lignin has been demonstrated to be catalyzed by laccase 15, a functional enzyme encoded by the AtTT10 gene. In this study, eight Brassica TT10 genes (three from B. napus, three from B. rapa and two from B. oleracea) were isolated and their roles in flavonoid oxidation/polymerization and lignin biosynthesis were investigated. Based on our phylogenetic analysis, these genes could be divided into two groups with obvious structural and functional differentiation. Expression studies showed that Brassica TT10 genes are active in developing seeds, but with differential expression patterns in yellow- and black-seeded near-isogenic lines. For functional analyses, three black-seeded B. napus cultivars were chosen for transgenic studies. Transgenic B. napus plants expressing antisense TT10 constructs exhibited retarded pigmentation in the seed coat. Chemical composition analysis revealed increased levels of soluble proanthocyanidins, and decreased extractable lignin in the seed coats of these transgenic plants compared with that of the controls. These findings indicate a role for the Brassica TT10 genes in proanthocyanidin polymerization and lignin biosynthesis, as well as seed coat pigmentation in B. napus

Genome-Wide Identification and Expression Analysis of nsLTP Gene Family in Rapeseed (Brassica napus) Reveals Their Critical Roles in Biotic and Abiotic Stress Responses

Non-specific lipid transfer proteins (nsLTPs) are small cysteine-rich basic proteins which play essential roles in plant growth, development and abiotic/biotic stress response. However, there is limited information about the nsLTP gene (BnLTP) family in rapeseed (Brassica napus). In this study, 283 BnLTP genes were identified in rapeseed, which were distributed randomly in 19 chromosomes of rapeseed. Phylogenetic analysis showed that BnLTP proteins were divided into seven groups. Exon/intron structure and MEME motifs both remained highly conserved in each BnLTP group. Segmental duplication and hybridization of rapeseed’s two sub-genomes mainly contributed to the expansion of the BnLTP gene family. Various potential cis-elements that respond to plant growth, development, biotic/abiotic stresses, and phytohormone signals existed in BnLTP gene promoters. Transcriptome analysis showed that BnLTP genes were expressed in various tissues/organs with different levels and were also involved in the response to heat, drought, NaCl, cold, IAA and ABA stresses, as well as the treatment of fungal pathogens (Sclerotinia sclerotiorum and Leptosphaeria maculans). The qRT-PCR assay validated the results of RNA-seq expression analysis of two top Sclerotinia-responsive BnLTP genes, BnLTP129 and BnLTP161. Moreover, batches of BnLTPs might be regulated by BnTT1 and BnbZIP67 to play roles in the development, metabolism or adaptability of the seed coat and embryo in rapeseed. This work provides an important basis for further functional study of the BnLTP genes in rapeseed quality improvement and stress resistance

MYB43 in Oilseed Rape (Brassica napus) Positively Regulates Vascular Lignification, Plant Morphology and Yield Potential but Negatively Affects Resistance to Sclerotinia sclerotiorum

Arabidopsis thaliana MYB43 (AtMYB43) is suggested to be involved in cell wall lignification. PtrMYB152, the Populus orthologue of AtMYB43, is a transcriptional activator of lignin biosynthesis and vessel wall deposition. In this research, MYB43 genes from Brassica napus (rapeseed) and its parental species B. rapa and B. oleracea were molecularly characterized, which were dominantly expressed in stem and other vascular organs and showed responsiveness to Sclerotinia sclerotiorum infection. The BnMYB43 family was silenced by RNAi, and the transgenic rapeseed lines showed retardation in growth and development with smaller organs, reduced lodging resistance, fewer silique number and lower yield potential. The thickness of the xylem layer decreased by 28%; the numbers of sclerenchymatous cells, vessels, interfascicular fibers, sieve tubes and pith cells in the whole cross section of the stem decreased by 28%, 59%, 48%, 34% and 21% in these lines, respectively. The contents of cellulose and lignin decreased by 17.49% and 16.21% respectively, while the pectin content increased by 71.92% in stems of RNAi lines. When inoculated with S. sclerotiorum, the lesion length was drastically decreased by 52.10% in the stems of transgenic plants compared with WT, implying great increase in disease resistance. Correspondingly, changes in the gene expression patterns of lignin biosynthesis, cellulose biosynthesis, pectin biosynthesis, cell cycle, SA- and JA-signals, and defensive pathways were in accordance with above phenotypic modifications. These results show that BnMYB43, being a growth-defense trade-off participant, positively regulates vascular lignification, plant morphology and yield potential, but negatively affects resistance to S. sclerotiorum. Moreover, this lignification activator influences cell biogenesis of both lignified and non-lignified tissues of the whole vascular organ

Detection of flavonoid composition in seed coats from transgenic and control <i>B. napus</i> plants.

<p>Analyses were performed by LC-UV-MS on seed coats of T₂ antisense <i>BnTT10</i> transgenic and control lines of <i>B. napus</i> cv. Zhongyou821. Q-3-G, Quercetin-3-glucoside; PC dimer B2, [DP2]-B2, epicatechin-(4β-8)-epicatechin; EC, epicatechin; K-3-O-G-7-O-G, kaempferol-3-O-glucoside-7-O-glucoside; I-di-H, isorhamnetin-dihexoside. Each value represents the means of three independent experiments +/− SD.</p

Lignin content in seed coats of transgenic and control lines.

<p>Lignin content was analyzed for seed coats of T₂ antisense <i>BnTT10</i> transgenic and control lines using the acetyl bromide method. Data are means for three T₂ progenies of each line, with triplicate measurements in each sample. The <i>P</i>-value is for a <i>t</i>-test for means of paired samples. W-10, W-12 and W-13: transgenic lines with inhibited <i>BnTT10</i> expression; W-22: transgenic lines with no inhibition in <i>BnTT10</i> expression; W-24: control lines with normal <i>BnTT10</i> expression. T₂-P: positive T₂ progenies; T₂-N: negative T₂ progenies after separation. Each value represents the means of three independent experiments +/− SD.</p

Chia (Salvia hispanica) experiment at a 30˚ N site in Sichuan Basin, China

ABSTRACT: The mysterious ancient Mesoamerican Indian crop chia (Salvia hispanica) is revived and expanding worldwide due to its richness of valuable nutraceuticals such as α-linolenic acid (ALA), antioxidants, food fiber, gels, and proteins. We carried out a pilot experiment on chia planting in non-frost Sichuan Basin, at Hechuan Base (30˚0′ 43″ N, 106˚7′ 41″ E, 216 m), Southwest University, Chongqing, China. The split-plot trial contained two factors, 3 spring-summer sowing times as main plots, and 6 densities as subplots, with 3 replicates. Phenological, botanical, adversity, yield, and seed quality traits were investigated. Plants were very tall, suffered from lodging, and flowered in mid-October. Sichuan Basin can be considered as a north edge for growing chia, with low yield (680 kg/hectare) because of insufficient seed filling and maturation in autumn-winter season (1000-seed weight of 1.14 g). However, its ALA content is 5 percent points higher than the seed-donor commercial bottle (65.06%/63.96% VS 59.35%/59.74% for black/white seeds), accompanied by decrease oleic and stearic acid, while linoleic acid and palmitic acid are equivalent. Considering its short-day habit, it is recommended to try sowing in middle summer (from late June to early August) to avoid too long growing period, excessive vegetative growth, and waste of field and climate resources caused by spring-summer sowing. Furthermore, winter sowing of chia with mulch cover could also be tried, with an expectation of harvesting in summer. Most importantly, only when the photoperiod-insensitive early flowering stocks are created, chia can be recommended as a low-risk crop to the farmers of this region

Seed pigmentation observation.

<p>Seedpods were sampled at 42, 50, 55, 60 and 45 DAF and the opened pods were observed under a low-power stereoscope. 5 DAH: five days after harvest. Seed coat pigmentation in the T₂ transgenic and control <i>B. napus</i> cv. Zhongyou821 (A) and Zhongshuang10 (B).</p

Soluble and insoluble PAs measured after acid-catalyzed hydrolysis in seed coats transgenic and control lines.

<p>The <i>P</i>-value is for a <i>t</i>-test for means of paired samples. W-10, W-12, W-13: transgenic lines with inhibited <i>BnTT10</i> expression; W-22: transgenic lines with no inhibition in <i>BnTT10</i> expression; W-24: control lines with normal <i>BnTT10</i> expression. T₂-P: positive T₂ progenies; T₂-N: negative T₂ progenies after separation. Soluble (A) and insoluble PA (B) content in seed coats of T₂ transgenic and control <i>B. napus</i> cv. Westar plants. Each value represents the means of three independent experiments +/− SD.</p