Light Intensity and Photoperiod Affect Growth and Nutritional Quality of Brassica Microgreens.

We explored the effects of different light intensities and photoperiods on the growth, nutritional quality and antioxidant properties of two Brassicaceae microgreens (cabbage Brassica oleracea L. and Chinese kale Brassica alboglabra Bailey). There were two experiments: (1) four photosynthetic photon flux densities (PPFD) of 30, 50, 70 or 90 µmoL·m-2·s-1 with red:blue:green = 1:1:1 light-emitting diodes (LEDs); (2) five photoperiods of 12, 14, 16, 18 or 20 h·d-1. With the increase of light intensity, the hypocotyl length of cabbage and Chinese kale microgreens shortened. PPFD of 90 µmol·m-2·s-1 was beneficial to improve the nutritional quality of cabbage microgreens, which had higher contents of chlorophyll, carotenoids, soluble sugar, soluble protein and vitamin C, as well as increased antioxidant capacity. The optimal PPFD for Chinese kale microgreens was 70 µmol·m-2·s-1. Increasing light intensity could increase the antioxidant capacity of cabbage and Chinese kale microgreens, while not significantly affecting glucosinolate (GS) content. The dry and fresh weight of cabbage and Chinese kale microgreens were maximized with a 14-h·d-1 photoperiod. The chlorophyll, carotenoid and soluble protein content in cabbage and Chinese kale microgreens were highest for a 16-h·d-1 photoperiod. The lowest total GS content was found in cabbage microgreens under a 12-h·d-1 photoperiod and in Chinese kale microgreens under 16-h·d-1 photoperiod. In conclusion, the photoperiod of 14~16 h·d-1, and 90 µmol·m-2·s-1 and 70 µmol·m-2·s-1 PPFD for cabbage and Chinese kale microgreens, respectively, were optimal for cultivation.

Phi thickenings in Brassica oleracea roots are induced by osmotic stress and mechanical effects, both involving jasmonic acid.

Phi thickenings are peculiar secondary cell wall thickenings found in radial walls of cortical cells in plant roots. However, while thickenings are widespread in the plant kingdom, research into their development has been lacking. Here, we describe a simple system for rapid induction of phi thickenings in primary roots of Brassica. Four-day-old seedlings were transferred from control agar plates to new plates containing increased levels of osmotica. Phi thickening development occurred within a narrow region of the differentiation zone proportional to osmolarity, with cellulose deposition and lignification starting after 12h and 15h, respectively. However, osmoprotectants not only failed to induce phi thickenings, but inhibited induction when tested in combination with thickening-inducing osmotica. An independent, biomechanical pathway exists regulating phi thickening induction, with root growth rates and substrate texture being important factors in determining thickening induction. Phi thickening development is also controlled by stress-related plant hormones, most notably jasmonic acid, but also abscisic acid. Our research not only provides the first understanding of the developmental pathways controlling phi thickening induction, but also provides tools with which the functions of these enigmatic structures might be clarified.

Demonstration of laser biospeckle method for speedy in vivo evaluation of plant-sound interactions with arugula.

In recent years, it is becoming clearer that plant growth and its yield are affected by sound with certain sounds, such as seedling of corn directing itself toward the sound source and its ability to distinguish stuttering of larvae from other sounds. However, methods investigating the effects of sound on plants either take a long time or are destructive. Here, we propose using laser biospeckle, a non-destructive and non-contact technique, to investigate the activities of an arugula plant for sounds of different frequencies, namely, 0 Hz or control, 100 Hz, 1 kHz, 10 kHz, including rock and classical music. Laser biospeckles are generated when scattered light from biological tissues interfere, and the intensities of such speckles change in time, and these changes reflect changes in the scattering structures within the biological tissue. A leaf was illuminated by light from a laser light of wavelength 635 nm, and the biospeckles were recorded as a movie by a CMOS camera for 20 sec at 15 frames per second (fps). The temporal correlation between the frames was characterized by a parameter called biospeckle activity (BA)under the exposure to different sound stimuli of classical and rock music and single-frequency sound stimuli for 1min. There was a clear difference in BA between the control and other frequencies with BA for 100 Hz being closer to control, while at higher frequencies, BA was much lower, indicating a dependence of the activity on the frequency. As BA is related to changes from both the surface as well as from the internal structures of the leaf, LSM (laser scanning microscope) observations conducted to confirm the change in the internal structure revealed more than 5% transient change in stomatal size following exposure to one minute to high frequency sound of 10kHz that reverted within ten minutes. Our results demonstrate the potential of laser biospeckle to speedily monitor in vivo response of plants to sound stimuli and thus could be a possible screening tool for selecting appropriate frequency sounds to enhance or delay the activity of plants. (337 words).

Identification of distinctive physiological and molecular responses to salt stress among tolerant and sensitive cultivars of broccoli (Brassica oleracea var. Italica).

BACKGROUND: Salt stress is one of the main constraints determining crop productivity, and therefore one of the main limitations for food production. The aim of this study was to characterize the salt stress response at the physiological and molecular level of different Broccoli (Brassica oleracea L. var. Italica Plenck) cultivars that were previously characterized in field and greenhouse trials as salt sensitive or salt tolerant. This study aimed to identify functional and molecular traits capable of predicting the ability of uncharacterized lines to cope with salt stress. For this purpose, this study measured different physiological parameters, hormones and metabolites under control and salt stress conditions. RESULTS: This study found significant differences among cultivars for stomatal conductance, transpiration, methionine, proline, threonine, abscisic acid, jasmonic acid and indolacetic acid. Salt tolerant cultivars were shown to accumulate less sodium and potassium in leaves and have a lower sodium to potassium ratio under salt stress. Analysis of primary metabolites indicated that salt tolerant cultivars have higher concentrations of several intermediates of the Krebs cycle and the substrates of some anaplerotic reactions. CONCLUSIONS: This study has found that the energetic status of the plant, the sodium extrusion and the proline content are the limiting factors for broccoli tolerance to salt stress. Our results establish physiological and molecular traits useful as distinctive markers to predict salt tolerance in Broccoli or to design novel biotechnological or breeding strategies for improving broccoli tolerance to salt stress.

Optimizing nutrient use efficiency, productivity, energetics, and economics of red cabbage following mineral fertilization and biopriming with compatible rhizosphere microbes.

Conventional agricultural practices and rising energy crisis create a question about the sustainability of the present-day food production system. Nutrient exhaustive crops can have a severe impact on native soil fertility by causing nutrient mining. In this backdrop, we conducted a comprehensive assessment of bio-priming intervention in red cabbage production considering nutrient uptake, the annual change in soil fertility, nutrient use efficiency, energy budgeting, and economic benefits for its sustainable intensification, among resource-poor farmers of Middle Gangetic Plains. The compatible microbial agents used in the study include Trichoderma harzianum, Pseudomonas fluorescens, and Bacillus subtilis. Field assays (2016-2017 and 2017-2018) of the present study revealed supplementing 75% of recommended NPK fertilizer with dual inoculation of T. harzianum and P. fluorescens increased macronutrient uptake (N, P, and K), root length, heading percentage, head diameter, head weight, and the total weight of red cabbage along with a positive annual change in soil organic carbon. Maximum positive annual change in available N and available P was recorded under 75% RDF + P. fluorescens + B. subtilis and 75% RDF + T. harzianum + B. subtilis, respectively. Bio-primed plants were also higher in terms of growth and nutrient use efficiency (agronomic efficiency, physiological efficiency, apparent recovery efficiency, partial factor productivity). Energy output (26,370 and 26,630 MJ ha-1), energy balance (13,643 and 13,903 MJ ha-1), maximum gross return (US $ 16,030 and 13,877 ha-1), and net return (US $ 15,966 and 13,813 ha-1) were considerably higher in T. harzianum, and P. fluorescens treated plants. The results suggest the significance of the bio-priming approach under existing integrated nutrient management strategies and the role of dual inoculations in producing synergistic effects on plant growth and maintaining the soil, food, and energy nexus.

Ectopic expression of a novel cold-resistance protein 1 from Brassica oleracea promotes tolerance to chilling stress in transgenic tomato.

Cold stress is considered as one of the major environmental factors that adversely affects the plant growth and distribution. Therefore, there arises an immediate need to cultivate effective strategies aimed at developing stress-tolerant crops that would boost the production and minimise the risks associated with cold stress. In this study, a novel cold-responsive protein1 (BoCRP1) isolated from Brassica oleracea was ectopically expressed in a cold susceptible tomato genotype Shalimar 1 and its function was investigated in response to chilling stress. BoCRP1 was constitutively expressed in all the tissues of B. oleracea including leaf, root and stem. However, its expression was found to be significantly increased in response to cold stress. Moreover, transgenic tomato plants expressing BoCRP1 exhibited increased tolerance to chilling stress (4 °C) with an overall improved rate of seed germination, increased root length, reduced membrane damage and increased accumulation of osmoprotectants. Furthermore, we observed increased transcript levels of stress responsive genes and enhanced accumulation of reactive oxygen species scavenging enzymes in transgenic plants on exposure to chilling stress. Taken together, these results strongly suggest that BoCRP1 is a promising candidate gene to improve the cold stress tolerance in tomato.

Doubled Haploid Broccoli (Brassica olearacea var. italica) Plants from Anther Culture.

Broccoli (Brassica olearecea var. italica) is a cole crop grown for its floral heads and stalks. It is rich in bioactive chemicals good for human health. Broccoli has been consumed as a vegetable since Roman times, but its production and consumption have increased significantly over the past few decades. Breeders try to develop new broccoli varieties with high yield, improved quality, and resistance to biotic and abiotic stresses. Almost all new broccoli varieties are F1 hybrids. Development of inbred broccoli lines that can be used as parents in hybrid production is a time-consuming and difficult process. Haploidization techniques can be utilized as a valuable support in broccoli breeding programs to speed up the production of genetically pure genotypes. Haploid plants of broccoli can be produced from immature male gametophytes via anther and microspore cultures with similar success rates. The most important parameters affecting the success of haploidization in broccoli are the genetic background (genotype) and the developmental stage of the microspores. Broccoli genotypes differ in their responses to androgenesis induction. The highest androgenesis response could be induced from microspores in late uninucleate and early binucleate stages. Recovery of diploid broccoli plants from haploids is possible via spontaneous and induced doubling. Doubled haploid (DH) broccoli lines are considered to be fully homozygous. Therefore, the production of DH lines is an alternative way to obtain pure inbred lines that can be utilized as parents in the development of new F1 hybrid varieties showing high levels of heterosis, high-quality heads, and uniform harvestable crop. We are using an anther culture-based haploid plant production system to develop DH broccoli lines in our broccoli breeding program. DH broccoli lines are produced from different genetic backgrounds within a year and handed to broccoli breeders.

Cabbage cultivars influence transfer and toxicity of cadmium in soil-Chinese flowering cabbage Brassica campestris-cutworm Spodoptera litura larvae.

We executed a pot experiment to examine the differences of absorption, chemical forms, subcellular distribution, and toxicity of Cd between two cultivars of Chinese flowering cabbage Brassica campestris [Lvbao701 (low-Cd cultivar) and Chicaixin No.4 (high-Cd cultivar)]. Compared to Chicaixin No.4, the presence of Lvbao701 enhanced the proportion of insoluble Cd forms in soil, Lvbao701 roots and leaves had higher proportion of Cd converted into insoluble phosphate precipitates and pectate-or protein-bound forms and lower proportion of inorganic Cd, which result in low accumulation and toxicity of Cd to Lvbao701 and cutworm Spodoptera litura fed on Lvbao701 leaves. Instead of total Cd, Cd transfer and toxicity in B. campestris-S. litura system depend on chemical Cd forms in soil and cabbages and subcellular Cd distributions in cabbages and insects, and the proportions of them were not the highest among all chemical forms and subcellular distributions of Cd. Although exchangeable Cd was major Cd chemical form in cabbage planted soil, Cd bound to iron and manganese oxides and to organic matter were significantly correlated with growth indices and photosynthesis parameters of cabbages. Despite major part of Cd was precipitated in cell wall of roots, Cd in organelle fraction was closely associated with the fitness of cabbages. Metal-rich granules, not cytosolic fraction (the major subcellular Cd distribution), affected the food utilization of S. litura. Therefore, cabbage cultivars significantly affected Cd transfer and toxicity in B. campestris-S. litura system, and the use of Lvbao701 in Cd polluted soil could reduce potential risks for Cd entering food chains.

Karrikinolide alleviates BDE-28, heat and Cd stressors in Brassica alboglabra by correlating and modulating biochemical attributes, antioxidative machinery and osmoregulators.

In this study, we have evaluated the role of karrikin (KAR1) against the absorption and translocation of a persistent organic pollutant (POP), 2,4,4'-Tribromodiphenyl ether (BDE-28) in plants, in the presence of two other stressors, cadmium (Cd) and high temperature. Furthermore, it correlates the physiological damages of Brassica alboglabra with the three stresssors separately. The results revealed that the post-germination application of KAR1 successfully augmented the growth (200%) and pertinent physiochemical parameters of B. alboglabra. KAR1 hindered air absorption of BDE-28 in plant tissues, and reduced its translocation coefficient (TF). Moreover, BDE-28 was the most negatively correlated (-0.9) stressor with chlorophyll contents, while the maximum mitigation by KAR1 was also achieved agaist BDE-28. The effect of temperature was more severe on soluble sugars (0.51), antioxidative machinery (-0.43), and osmoregulators (0.24). Cd exhibited a stronger inverse interrelation with the enzymatic antioxidant cascade. Application of KAR1 mitigated the deleterious effects of Cd and temperature stress on plant physiological parameters along with reduced aero-concentration factor, TF, and metal tolerance index. The phytohormone reduced lipid peroxidation by decreasing synthesis of ROS and persuading its breakdown. The stability of cellular membranes was perhaps due to the commotion of KAR1 as a growth-promoting phytohormone. In the same way, KAR1 supplementation augmented the membrane stability index, antioxidant defense factors, and removal efficiency of the pollutants. Consequently, the exogenously applied KAR1 can efficiently alleviate Cd stress, heat stress, and POP toxicity.

Jasmonic acid-mediated enhanced regulation of oxidative, glyoxalase defense system and reduced chromium uptake contributes to alleviation of chromium (VI) toxicity in choysum (Brassica parachinensis L.).

The cultivation of leafy vegetables on metal contaminated soil embodies a serious threat to yield and quality. In the present study, the potential role of exogenous jasmonic acid (JA; 0, 5, 10, and 20 µM) on mitigating chromium toxicity (Cr; 0, 150, and 300 µM) was investigated in choysum (Brassica parachinensis L.). With exposure to increasing Cr stress levels, a dose-dependent decline in growth, photosynthesis, and physio-biochemical attributes of choysum plants was observed. An increase in Cr levels also resulted in oxidative stress closely associated with higher lipoxygenase activity (LOX), hydrogen peroxide (H2O2) generation, lipid peroxidation (MDA), and methylglyoxal (MG) levels. Exogenous application of JA alleviated the Cr-induced phytotoxic effects on photosynthetic pigments, gas exchange parameters, and restored growth of choysum plants. While exposed to Cr stress, JA supplementation induced plant defense system via enhanced regulation of antioxidant enzymes, ascorbate and glutathione pool, and the glyoxalase system enzymes. The coordinated regulation of antioxidant and glyoxalase systems expressively suppressed the oxidative and carbonyl stress at both Cr stress levels. More importantly, JA restored the mineral nutrient contents, restricted Cr uptake, and accumulation in roots and shoots of choysum plants when compared to the only Cr-stressed plants. Overall, the application of JA2 treatment (10 µM JA) was more effective and counteracted the detrimental effects of 150 µM Cr stress by restoring the growth and physio-biochemical attributes to the level of control plants, while partially mitigated the detrimental effects of 300 µM Cr stress. Hence, JA application might be considered as an effective approach for minimizing Cr uptake and its detrimental effects in choysum plants grown on contaminated soils.

Development of Ogura CMS restorers in Brassica oleracea subspecies via direct RfoB gene transformation.

KEY MESSAGE: The Ogura CMS RfoB restorer developing via RfoB gene transformation was utilized to produce specific morphological Ogura CMS restorers and clubroot resistance lines in Brassica oleracea subspecies. Brassica oleracea vegetables including cabbage, cauliflower, kohlrabi, Brussels sprouts and Chinese kale are morphologically very different despite being members of the same species. The Ogura cytoplasmic male sterility (CMS) system is the most stable strategy for the hybrid breeding of these species. However, this limits the utilization of some excellent genes due to the lack of fertile restorer genes in the system. Herein, to efficaciously use Ogura CMS, the Ogura CMS RfoB restorer was produced by transforming the modified RfoB restorer gene into the Ogura CMS line 'CMS2016' of B. oleracea var. capitata. This gene was shown to recover fertility of natural Ogura CMS lines in B. oleracea subspecies and create transient Ogura CMS RfoB restorers such as the clubroot resistance Ogura CMS RfoB restorer. Interestingly, clubroot resistant individuals without transgenic elements were screened in the progenies of hybrids between B. oleracea inbred lines and the clubroot resistance Ogura CMS RfoB restorer. In addition, 18 different morphological Ogura CMS restorers were developed to specifically recover fertile of Ogura CMS cultivars in B. oleracea subspecies.

Genome-wide identification and analysis of NPR family genes in Brassica juncea var. tumida.

Nonexpressor of pathogenesis-related (NPR) genes are bona fide transcription cofactors in the signal transduction pathway of salicylic acid (SA) and play critical regulatory roles in plant immunity. However, the NPR family genes in Brassica juncea var. tumida have not yet been comprehensively identified and analyzed as of yet. In the present study, NPR genes in B. juncea var. tumida seedlings were identified, and the tissue-specific expression patterns of NPR genes in the seedling were analyzed under salt stress (200 mM) treatment and infection by Plasmodiophora brassicae. A total of 19 NPR family genes clustering into six separate groups were identified in the genome of B. juncea var. tumida. These BjuNPR family genes were located in 11 of 18 chromosomes of B. juncea var. tumida and each possessed 1-5 exons. The BjuNPR family members had similar protein structures and conserved motifs. The BjuNPR genes exhibited tissue-specific expression patterns in the root, stem, leaf, flower and pod. Some BjuNPR genes were sensitive to salt stress and showed up-regulated or down-regulated expression patterns and most BjuNPR genes were up-regulated upon infection by P. brassicae. This study provides a foundation for further research into BjuNPR genes regulation in plant growth, development, and abiotic stress tolerance.

Targeting salt stress coping mechanisms for stress tolerance in Brassica: A research perspective.

Brassica genus comprises numerous cultivated brassica species with various economic importance. Salt stress is an overwhelming problem causing serious losses in Brassica species (e.g. B. napus, B. rapa, B. oleracea, B. juncea) growth and grain yield production by inducing ionic and ROS toxicity. Given that a significant variation exists in salt tolerance level in Brassica genus, Brassica species exhibited numerous salt tolerance mechanisms which were either overlooked or given less importance to improve and understand innate salt stress tolerance mechanism in Brassica species. In this review, we tried to highlight the importance and recent findings relating to some overlooked and potential mechanisms such as role of neurotransmitters, and role of cytosolic Ca2+ and ROS as signaling elements to enhance salt stress tolerance. Studies revealed that salt tolerant brassica species retained more K+ in leaf mesophyll which confers overall salinity tolerance in salt tolerance brassica species. Neurotransmitter such as melatonin, dopamiane and eATP regulates K+ and Ca2+ permeable ion channels and plays a very crucial role in ionic homeostasis under salinity stress in brassica. At the end, the numerous possible salt stress agronomic strategies were also discussed to mitigate the severity of the salt stress in Brassica species.

Effect of folic acid on the postharvest physiology of broccoli during storage.

The objective of the present study was to explore the effect of folic acid on the postharvest physiology of broccoli placed in storage. Broccoli heads were immersed in 5 mg L-1 folic acid for 10 min, then stored at 20 ± 1 °C for 4 days. Results indicated that the postharvest treatment of broccoli with folic acid decreased the rate of flower opening and yellowing, inhibited weight loss, reduced the level of respiration, as well as ethylene generation. Folic acid-treated broccoli maintained their level of chlorophyll, total soluble solids, vitamin C, total phenolics, flavonoids, glucosinolate, and folic acid. Treated broccoli also exhibited reduced accumulation of malondialdehyde (MDA) and reactive oxygen species (ROS). Concomitantly, antioxidant enzyme activity and corresponding gene expression were also enhanced. In contrast, chlorophyll-degrading enzyme gene expression was suppressed. These results indicated that folic acid treatment of broccoli could be used to prolong shelf-life.

Gene duplication and stress genomics in Brassicas: Current understanding and future prospects.

Polyploidy or whole genome duplication (WGD) is an evolutionary phenomenon that happened in all angiosperms multiple times over millions of years. Extensive studies on the model plant Arabidopsis thaliana genome have revealed that it has undergone five rounds of WGDs followed, in the Brassicaceae tribe, by a characteristic whole genome triplication (WGT). In addition, small-scale events such as tandem or segmental duplications and retrotransposition also enable plants to reshape their genomes. Over the decades, extensive research efforts have been undertaken to understand the evolutionary significance of polyploidy. On the other hand, much less attention has been paid to understanding the impact of gene duplication on the diversification of important stress response genes. The main objective of this review is to discuss key aspects of gene and genome duplications with a focus on genes primarily regulated by osmotic stresses. The focal family is the Brassicaceae, since it (i) underwent multiple rounds of WGDs plus WGTs, (ii) hosts many economically important crops and wild relatives that are tolerant to a range of stresses, and (iii) comprises many species that have already been sequenced. Diverse molecular mechanisms that lead to structural and regulatory alterations of duplicated genes are discussed. Examples are drawn from recent literature to elucidate expanded, stress responsive gene families identified from different Brassica crops. A combined bioinformatic and transcriptomic method has been proposed and tested on a known stress-responsive gene pair to prove that stress-responsive duplicated allelic variants can be identified by this method. Finally, future prospects for engineering these genes into crops to enhance stress tolerance are discussed, and important resources for Brassica genome research are provided.

Overexpression of Two CCCH-type Zinc-Finger Protein Genes Leads to Pollen Abortion in Brassica campestris ssp. chinensis.

The pollen grains produced by flowering plants are vital for sexual reproduction. Previous studies have shown that two CCCH-type zinc-finger protein genes in Brassica campestris, BcMF30a and BcMF30c, are involved in pollen development. Due to their possible functional redundancy, gain-of-function analysis is helpful to reveal their respective biological functions. Here, we found that the phenotypes of BcMF30a and BcMF30c overexpression transgenic plants driven by their native promoters were similar, suggesting their functional redundancy. The results showed that the vegetative growth was not affected in both transgenic plants, but male fertility was reduced. Further analysis found that the abortion of transgenic pollen was caused by the degradation of pollen contents from the late uninucleate microspore stage. Subcellular localization analysis demonstrated that BcMF30a and BcMF30c could localize in cytoplasmic foci. Combined with the studies of other CCCH-type genes, we speculated that the overexpression of these genes can induce the continuous assembly of abnormal cytoplasmic foci, thus resulting in defective plant growth and development, which, in this study, led to pollen abortion. Both the overexpression and knockout of BcMF30a and BcMF30c lead to abnormal pollen development, indicating that the appropriate expression levels of these two genes are critical for the maintenance of normal pollen development.

Response of wheat, pea, and canola to micronutrient fertilization on five contrasting prairie soils.

A polyhouse study was conducted to evaluate the relative effectiveness of different micronutrient fertilizer formulation and application methods on wheat, pea and canola, as indicated by yield response and fate of micronutrients in contrasting mineral soils. The underlying factors controlling micronutrient bioavailability in a soil-plant system were examined using chemical and spectroscopic speciation techniques. Application of Cu significantly improved grain and straw biomass yields of wheat on two of the five soils (Ukalta and Sceptre), of which the Ukalta soil was critically Cu deficient according to soil extraction with DTPA. The deficiency problem was corrected by either soil or foliar application of Cu fertilizers. There were no significant yield responses of pea to Zn fertilization on any of the five soils. For canola, soil placement of boric acid was effective in correcting the deficiency problem in Whitefox soil, while foliar application was not. Soil extractable Cu, Zn, and B concentration in post-harvest soils were increased with soil placement of fertilizers, indicating that following crops in rotation could benefit from this application method. The chemical and XANES spectroscopic speciation indicates that carbonate associated is the dominant form of Cu and Zn in prairie soils, where chemisorption to carbonates is likely the major process that determines the fate of added Cu and Zn fertilizer.

Natural variation in glycine-rich region of Brassica oleracea cold shock domain protein 5 (BoCSDP5) is associated with low temperature tolerance.

BACKGROUND: Low temperature (LT) or cold stress is a major environmental stress that seriously affects plant growth and development, limiting crop productivity. Cold shock domain proteins (CSDPs), which are present in most living organism, are involved in RNA metabolisms influencing abiotic stress tolerance. OBJECTIVE: The aims of this study are to identify target gene for LT-tolerance, like CSDPs, characterize genetics, and develop molecular marker distinguishing LT-tolerance in cabbage (Brassica oleracea var. capitata). METHODS: Semi-quantitative RT-PCR or qRT-PCR was used in gene expression study. LT-tolerance was determined by electrolyte leakage and PCR with allelic specific primers. RESULTS: Allelic variation was found in BoCSDP5 coding sequence (CDs) between LT-tolerant (BN106 and BN553) and -susceptible inbred lines (BN107 and BN554). LT-tolerant inbred lines contained variant type of BoCSDP5 (named as BoCSDP5v) which encodes extra CCHC zinc finger domain at C-terminus. Association of LT-tolerance with BoCSDP5v was confirmed by electrolyte leakage and segregation using genetic population derived from BN553 and BN554 cross. Allelic variation in BoCSDP5 gene does not influence the rate of gene expression, but produces different proteins with different number of CCHC zinc finger domains. LT-tolerance marker designed on the basis of polymorphism between BoCSDP5 and BoCSDP5v was confirmed with samples used in previous B. oleracea CIRCADIAN CLOCK ASSOCIATED 1 (BoCCA1) marker validation. CONCLUSIONS: LT-tolerant allele (BoCSDP5v) is dominant and independent of CBF pathway, and sufficient to generate molecular markers to identify LT-tolerant cabbage when it is used in combination with another marker, like BoCCA1-derived one. Production and analysis of overexpressing plants of BoCSDP1, BoCSDP3, BoCSDP5 and BoCSDP5v will be required for elucidating the function of CCHC zinc finger domains in LT-tolerance.

Detergent Resistant Membrane Domains in Broccoli Plasma Membrane Associated to the Response to Salinity Stress.

Detergent-resistant membranes (DRMs) microdomains, or "raft lipids", are key components of the plasma membrane (PM), being involved in membrane trafficking, signal transduction, cell wall metabolism or endocytosis. Proteins imbibed in these domains play important roles in these cellular functions, but there are few studies concerning DRMs under abiotic stress. In this work, we determine DRMs from the PM of broccoli roots, the lipid and protein content, the vesicles structure, their water osmotic permeability and a proteomic characterization focused mainly in aquaporin isoforms under salinity (80 mM NaCl). Based on biochemical lipid composition, higher fatty acid saturation and enriched sterol content under stress resulted in membranes, which decreased osmotic water permeability with regard to other PM vesicles, but this permeability was maintained under control and saline conditions; this maintenance may be related to a lower amount of total PIP1 and PIP2. Selective aquaporin isoforms related to the stress response such as PIP1;2 and PIP2;7 were found in DRMs and this protein partitioning may act as a mechanism to regulate aquaporins involved in the response to salt stress. Other proteins related to protein synthesis, metabolism and energy were identified in DRMs independently of the treatment, indicating their preference to organize in DMRs.

Complexity of Brassica oleracea-Alternaria brassicicola Susceptible Interaction Reveals Downregulation of Photosynthesis at Ultrastructural, Transcriptional, and Physiological Levels.

Black spot disease, caused by Alternaria brassicicola in Brassica species, is one of the most devastating diseases all over the world, especially since there is no known fully resistant Brassica cultivar. In this study, the visualization of black spot disease development on Brassica oleracea var. capitata f. alba (white cabbage) leaves and subsequent ultrastructural, molecular and physiological investigations were conducted. Inter- and intracellular hyphae growth within leaf tissues led to the loss of host cell integrity and various levels of organelle disintegration. Severe symptoms of chloroplast damage included the degeneration of chloroplast envelope and grana, and the loss of electron denseness by stroma at the advanced stage of infection. Transcriptional profiling of infected leaves revealed that photosynthesis was the most negatively regulated biological process. However, in infected leaves, chlorophyll and carotenoid content did not decrease until 48 hpi, and several chlorophyll a fluorescence parameters, such as photosystem II quantum yield (Fv/Fm), non-photochemical quenching (NPQ), or plant vitality parameter (Rdf) decreased significantly at 24 and 48 hpi compared to control leaves. Our results indicate that the initial stages of interaction between B. oleracea and A. brassicicola are not uniform within an inoculation site and show a complexity of host responses and fungal attempts to overcome host cell defense mechanisms. The downregulation of photosynthesis at the early stage of this susceptible interaction suggests that it may be a part of a host defense strategy, or, alternatively, that chloroplasts are targets for the unknown virulence factor(s) of A. brassicicola. However, the observed decrease of photosynthetic efficiency at the later stages of infection is a result of the fungus-induced necrotic lesion expansion.