Frequent gain- and loss-of-function mutations of the BjMYB113 gene accounted for leaf color variation in Brassica juncea.

BACKGROUND: Mustard (Brassica juncea) is an important economic vegetable, and some cultivars have purple leaves and accumulate more anthocyanins than the green. The genetic and evolution of purple trait in mustard has not been well studied. RESULT: In this study, free-hand sections and metabolomics showed that the purple leaves of mustard accumulated more anthocyanins than green ones. The gene controlling purple leaves in mustard, Mustard Purple Leaves (MPL), was genetically mapped and a MYB113-like homolog was identified as the candidate gene. We identified three alleles of the MYB113-like gene, BjMYB113a from a purple cultivar, BjMYB113b and BjMYB113c from green cultivars. A total of 45 single nucleotide polymorphisms (SNPs) and 8 InDels were found between the promoter sequences of the purple allele BjMYB113a and the green allele BjMYB113b. On the other hand, the only sequence variation between the purple allele BjMYB113a and the green allele BjMYB113c is an insertion of 1,033-bp fragment in the 3'region of BjMYB113c. Transgenic assay and promoter activity studies showed that the polymorphism in the promoter region was responsible for the up-regulation of the purple allele BjMYB113a and high accumulation of anthocyanin in the purple cultivar. The up-regulation of BjMYB113a increased the expression of genes in the anthocyanin biosynthesis pathway including BjCHS, BjF3H, BjF3'H, BjDFR, BjANS and BjUGFT, and consequently led to high accumulation of anthocyanin. However, the up-regulation of BjMYB113 was compromised by the insertion of 1,033-bp in 3'region of the allele BjMYB113c. CONCLUSIONS: Our results contribute to a better understanding of the genetics and evolution of the BjMYB113 gene controlling purple leaves and provide useful information for further breeding programs of mustard.

Heterotrimeric Gα subunit regulates plant architecture, organ size and seed weight in the oilseed Brassica juncea.

KEY MESSAGE: Two BjuGα proteins exhibit conserved GTP-binding and GTP-hydrolysis activities, and function in maintaining overall plant architecture and controlling multiple yield-related traits in the oilseed Brassica juncea. Heterotrimeric G-protein (Gα, Gß and Gγ) are key signal transducers, well characterized in model plants Arabidopsis and rice. However, our knowledge about the roles played by G-proteins in regulating various growth and developmental traits in polyploid crops, having a complex G-protein signalling network, is quite sparse. In the present study, two Gα encoding genes (BjuA.Gα1 and BjuB.Gα1) were isolated from the allotetraploid Brassica juncea, a globally cultivated oilseed crop of the Brassicaceae family. BjuGα1 genes share a close evolutionary relationship, and the encoded proteins exhibit highly conserved G-protein activities while showing expression differentiation, wherein BjuA.Gα1 was the highly abundant transcript during plant growth and developmental stages. RNAi based suppression of BjuGα1 displayed compromised effects on most of the tested vegetative and reproductive parameters, particularly plant height (32-58%), flower and siliques dimensions, and seed weight (11-13%). Further, over-expression of a constitutively active Gα, lacking the GTPase activity, produced plants with increased height, organ size and seed weight (7-25%), without altering seed quality traits like fatty acid composition, glucosinolates, oil and protein contents. Our study demonstrates that BjuGα1 proteins control overall plant architecture and multiple yield-related traits in the oilseed B. juncea, suggesting that BjuGα1 could be a promising target for crop improvement.

Elucidating the interaction between light competition and herbivore feeding patterns using functional-structural plant modelling.

Background and Aims: Plants usually compete with neighbouring plants for resources such as light as well as defend themselves against herbivorous insects. This requires investment of limiting resources, resulting in optimal resource distribution patterns and trade-offs between growth- and defence-related traits. A plant's competitive success is determined by the spatial distribution of its resources in the canopy. The spatial distribution of herbivory in the canopy in turn differs between herbivore species as the level of herbivore specialization determines their response to the distribution of resources and defences in the canopy. Here, we investigated to what extent competition for light affects plant susceptibility to herbivores with different feeding preferences. Methods: To quantify interactions between herbivory and competition, we developed and evaluated a 3-D spatially explicit functional-structural plant model for Brassica nigra that mechanistically simulates competition in a dynamic light environment, and also explicitly models leaf area removal by herbivores with different feeding preferences. With this novel approach, we can quantitatively explore the extent to which herbivore feeding location and light competition interact in their effect on plant performance. Key Results: Our results indicate that there is indeed a strong interaction between levels of plant-plant competition and herbivore feeding preference. When plants did not compete, herbivory had relatively small effects irrespective of feeding preference. Conversely, when plants competed, herbivores with a preference for young leaves had a strong negative effect on the competitiveness and subsequent performance of the plant, whereas herbivores with a preference for old leaves did not. Conclusions: Our study predicts how plant susceptibility to herbivory depends on the composition of the herbivore community and the level of plant competition, and highlights the importance of considering the full range of dynamics in plant-plant-herbivore interactions.

Cadmium toxicity-induced proline accumulation is coupled to iron depletion.

Investigations were conducted to elucidate the key factor behind Cd2+-toxicity-induced proline accumulation in Indian mustard (Brassica juncea) by raising seedlings, independently in distilled water (DW) and mineral growth medium (MGM) in the presence of 0-500 µM CdCl2. Invariably, Cd2+-induced toxicity, measured in terms of growth, was significantly more prominent in seedlings raised in DW than those raised in MGM. Cd2+ brought about a significant reduction in growth and photosystem II activity with a concomitant increase in proline levels, in a concentration-dependent manner. Interestingly, the level of iron in shoots of seedlings decreased proportionately with increase in Cd2+ toxicity. Cd2+-promoted proline accumulation was significantly higher in seedlings raised in DW than those raised in MGM. Depletion of essential cations (viz. Ca2+, Mg2+, K+, and Fe2+) from MGM one at a time revealed that depletion of Fe2+ leads to maximal proline accumulation under Cd2+ toxicity. Interestingly, proline level in seedlings raised under Cd2+ toxicity in DW supplemented with Fe2+ was similar to that recorded in seedlings raised in MGM. Our results convincingly demonstrated that Cd2+-induced iron deficiency promotes proline accumulation.

Heritability and Reversibility of DNA Methylation Induced by in vitro Grafting between Brassica juncea and B. oleracea.

Grafting between tuber mustard and red cabbage produced a chimeric shoot apical meristem (SAM) of TTC, consisting of Layers I and II from Tuber mustard and Layer III from red Cabbage. Phenotypic variations, which mainly showed in leaf shape and SAM, were observed in selfed progenies GSn (GS = grafting-selfing, n = generations) of TTC. Here the heritability of phenotypic variation and its association with DNA methylation changes in GSn were investigated. Variation in leaf shape was found to be stably inherited to GS5, but SAM variation reverted over generations. Subsequent measurement of DNA methylation in GS1 revealed 5.29-6.59% methylation changes compared with tuber mustard (TTT), and 31.58% of these changes were stably transmitted to GS5, but the remainder reverted to the original status over generations, suggesting grafting-induced DNA methylation changes could be both heritable and reversible. Sequence analysis of differentially methylated fragments (DMFs) revealed methylation mainly changed within transposons and exon regions, which further affected the expression of genes, including flowering time- and gibberellin response-related genes. Interestingly, DMFs could match differentially expressed siRNA of GS1, GS3 and GS5, indicating that grafting-induced DNA methylation could be directed by siRNA changes. These results suggest grafting-induced DNA methylation may contribute to phenotypic variations induced by grafting.

Physiological and morphological responses of the root system of Indian mustard (Brassica juncea L. Czern.) and rapeseed (Brassica napus L.) to copper stress.

Copper (Cu) is an essential microelement for growth and development, but in excess it can cause toxicity in plants. In this comparative study, the uptake and accumulation of Cu as well as the morphological and physiological responses of Indian mustard (Brassica juncea L. Czern.) and rapeseed (Brassica napus L.) roots to Cu treatment were investigated. The possible involvement of redox active molecules (reactive oxygen species and nitric oxide) and modification in cell wall structure associated with Cu-induced morphological responses were also studied. In short- and long-term treatments, B. juncea suffered more pronounced growth inhibition as compared with B. napus. In addition to the shortening of primary and lateral roots, the number and the density of the laterals were also decreased by Cu. Exposure to copper induced nitric oxide generation in the root tips and this event proved to be dependent on the duration of the exposure and on the plant species. In short- and long-term treatments, Indian mustard showed more significant activation of superoxide dismutase (SOD), inhibition of ascorbate peroxidase (APX) and oxidation of ascorbate (AsA) than B. napus. Moreover, H2O2-dependent lignification was also observed in the Cu-exposed plants. In longer term, significant AsA accumulation and callose deposition were observed, reflecting serious oxidative stress in B. juncea. Based on the morphological and physiological results, we conclude that rapeseed tolerates Cu excess better than Indian mustard.

A novel indirect defence in Brassicaceae: structure and function of extrafloral nectaries in Brassica juncea.

While nectaries are commonly found in flowers, some plants also form extrafloral nectaries on stems or leaves. For the first time in the family Brassicaceae, here we report extrafloral nectaries in Brassica juncea. The extrafloral nectar (EFN) was secreted from previously amorphic sites on stems, flowering stalks and leaf axils from the onset of flowering until silique formation. Transverse sections at the point of nectar secretion revealed a pocket-like structure whose opening was surrounded by modified stomatal guard cells. The EFN droplets were viscous and up to 50% of the total weight was sugars, 97% of which was sucrose in the five varieties of B. juncea examined. Threonine, glutamine, arginine and glutamate were the most abundant amino acids. EFN droplets also contained glucosinolates, mainly gluconapin and sinigrin. Nectar secretion was increased when the plants were damaged by chewing above- and belowground herbivores and sap-sucking aphids. Parasitoids of each herbivore species were tested for their preference, of which three parasitoids preferred EFN and sucrose solutions over water. Moreover, the survival and fecundity of parasitoids were positively affected by feeding on EFN. We conclude that EFN production in B. juncea may contribute to the indirect defence of this plant species.

The effects of seed size on hybrids formed between oilseed rape (Brassica napus) and wild brown mustard (B. juncea).

BACKGROUND: Seed size has significant implications in ecology, because of its effects on plant fitness. The hybrid seeds that result from crosses between crops and their wild relatives are often small, and the consequences of this have been poorly investigated. Here we report on plant performance of hybrid and its parental transgenic oilseed rape (Brassica napus) and wild B. juncea, all grown from seeds sorted into three seed-size categories. METHODOLOGY/PRINCIPAL FINDINGS: Three seed-size categories were sorted by seed diameter for transgenic B. napus, wild B. juncea and their transgenic and non-transgenic hybrids. The seeds were sown in a field at various plant densities. Globally, small-seeded plants had delayed flowering, lower biomass, fewer flowers and seeds, and a lower thousand-seed weight. The seed-size effect varied among plant types but was not affected by plant density. There was no negative effect of seed size in hybrids, but it was correlated with reduced growth for both parents. CONCLUSIONS: Our results imply that the risk of further gene flow would probably not be mitigated by the small size of transgenic hybrid seeds. No fitness cost was detected to be associated with the Bt-transgene in this study.

In vitro biochemical evaluation of cadmium tolerance mechanism in callus and seedlings of Brassica juncea.

In vitro grown callus and seedlings of Brassica juncea were treated with equimolar concentrations of cadmium and compared for their respective tolerance to cadmium. Calli cultures were grown on Murashige and Skoog medium supplemented with alpha 6-benzyl aminopurine (200 microg (-1), naphthalene acetic acid 200 microg L(-1)) and 2,4-dichloro-phenoxy acetic acid (65 microg L(-1)) while the seedlings grown on Hoagland's nutrient solution have been carried out. Cellular homeostasis and detoxification to cadmium in B. juncea were studied by analyzing the growth in terms of fresh weight and dry weight, lipid peroxidation, proline accumulation, and antioxidative enzymes (superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT)). At 200 microM cadmium, callus and seedlings showed 73.61% and 74.76% reduction in tolerance, respectively. A significant increase in malondialdehyde (MDA) content was found in both calli and seedlings; however, the amount of MDA content was more in seedlings. Proline content increased on lower concentration of cadmium (up to 50 microM), and it further decreased (up to 200 microM). But the accumulation of proline was higher in callus cultures. The overall activity of antioxidative enzymes (SOD, CAT, and APX) was found to be higher in callus in comparison to seedlings of B. juncea. Callus and seedlings showed a significant (P

Separation and determination of heavy metals associated with low molecular weight chelators in xylem saps of Indian mustard (Brassica juncea) by size exclusion chromatography and atomic absorption spectrometry.

To elucidate the role of low molecular weight chelators in long-distance root-to-shoot transport of heavy metals in Indian mustard, an "off-line" size exclusion high-performance liquid chromatography-graphite furnace atomic absorption spectrometry was developed to investigate heavy metals associated with low molecular weight chelators in xylem saps of Indian mustard (Brassica juncea). The size exclusion chromatogram presented only the peaks with molecular weight for all xylem saps and directly indicated the long-distance transport of phytochelatins (PCs) of Indian mustard under Cd stress. In the absence of Cd stress, only organic acids and inorganic anions participated in the long-distance transport of Cd, but organic acids, inorganic anions, glutathione (GSH), and cysteine might relate to the long-distance transport of Cu or Zn. In the presence of Cd stress, PCs were induced, and Cd ions in xylem saps were associated with the induced PCs. As the Cd levels in nutrient solution increased, more Cd in xylem saps adopted the form of PC-Cd. Although PCs might participate in the long-distance transport of Cd under Cd stress, the majority of Cd was still transported by organic acids and inorganic anions in xylem vessels. Moreover, results indicated the existence of complexation competition for GSH and cysteine between Cd and Cu (or Zn) and complexation competition for Cd between PCs and GSH (or cysteine) in xylem vessels. Our work might be very useful for understanding the mechanism of long-distance transport of heavy metals in hyperaccumulator.

A new cytoplasmic male sterility system for hybrid seed production in Indian oilseed mustard Brassica juncea.

We report a novel cytoplasmic male sterility (CMS) system in Brassica juncea (oilseed mustard) which could be used for production of hybrid seed in the crop. A male sterile plant identified in a microspore derived doubled haploid population of re-synthesized B. napus line ISN 706 was found to be a CMS as the trait was inherited from the female parent. This CMS, designated '126-1', was subsequently transferred to ten different B. juncea varieties and lines through inter-specific crosses followed by recurrent backcrossing. The F(1)s of inter-specific crosses were invariably partially fertile, but irrespective of the variety/line used, the recipient lines became progressively male sterile over five to seven generations and could be maintained by crossing the male sterile lines with their normal counterparts. The male sterile lines were found to be stable for the trait under both long and short day conditions. CMS lines when crossed with lines other than the respective maintainer line were restored for fertility, implying that any variety could act as a restorer for '126-1' cytoplasm in B. juncea. These unique features in maintenance and restoration of CMS lines coupled with near normal floral morphology of the CMS lines have allowed the use of '126-1' cytoplasm for hybrid seed production. The uniqueness of '126-1' has been further established by Southern hybridization with mitochondrial DNA probes and by a histological study of the development of male sterile anthers.

Effect of scale of Cd heterogeneity and timing of exposure on the Cd uptake and shoot biomass, of plants with a contrasting root morphology.

A pot experiment was conducted to investigate the influence of spatial heterogeneity of Cd distribution in soil on shoot biomass, shoot metal concentration and total shoot Cd uptake by lettuce (Lactuca sativa, variety Tom Thumb) and Indian mustard (Brassica juncea). Five different soil treatments had similar overall concentration of Cd per pot, but different scales of heterogeneity and also timing of plant exposure during the growth cycle. The presence and scale of heterogeneity and timing of exposure were found to have significant effects on shoot biomass for both plants (with one exception). The mean values of Cd mass taken up were significantly affected by the presence of heterogeneity and timing only for lettuce. Only the scale of heterogeneity affected the uptake of Cd by Indian mustard, presumably because of its larger root system (approximately 18 cm, compared with approximately 5 cm for lettuce). These findings have important implications for phytoremediation, and for human health risk assessment where leafy vegetables are grown in situations with highly elevated Cd concentrations.