Evidence of glucosinolates translocation from inflorescences to stems during postharvest storage of broccoli.

Broccoli is a vegetable appreciated by consumers for its nutritional properties, particularly for its high glucosinolate (GLS) content. However, broccoli shows a high rate of senescence during postharvest and the GLS content in inflorescences decreases sharply. Usually, postharvest studies on broccoli focus on inflorescences, ignoring the other tissues harvested such as the stems and main stalk. In this work, GLS metabolism in whole heads of broccoli (including inflorescences, small stems and stalk) was analysed during postharvest senescence. The content of GLS content, expression of GLS metabolic genes, and expression of GLS transport-associated genes were measured in the three parts of harvested broccoli. A marked decrease in the content of all GLSs was detected in inflorescences, but an increase in the stems and stalk. Also, decreased expressions of GLS biosynthesis and degradation genes were detected in all tissues analysed. On the other hand, an increase in the expression of one of the genes involved in GLS transport was observed. These results suggest that GLSs would be transported from inflorescences to stems during postharvest senescence. From a commercial point of view, broccoli stems are usually discarded and not used as food. However, the accumulation of GLSs in the stems is an important factor to consider when contemplating potential commercial use of this part of the plant.

Phenylalanine ammonia lyase is more relevant than Chalcone synthase and Chalcone isomerase in the biosynthesis of flavonoids during postharvest senescence of broccoli.

Broccoli contains a high content of nutraceutical compounds, such as glucosinolates and flavonoids. In this work, the effect of different treatments that modulate postharvest senescence of broccoli was evaluated and flavonoid metabolism during postharvest storage was analyzed at 20°C. A decrease in hue angle (HUE°) and chlorophylls and an increase in flavonoid content were detected during senescence. It observed that most of the treatments that delayed senescence also decreased flavonoid content, except visible light and UV-C treatments. In all cases, a direct correlation between those treatments that increased flavonoid biosynthesis and BoPAL gene expression was detected. This response was not detected in the expression of the other two flavonoid synthesis relevant genes BoCHS and BoCHI, suggesting that BoPAL has a greater influence on the regulation of the via, during broccoli senescence. PRACTICAL APPLICATIONS: Broccoli is a vegetable with valuable nutritional properties. Because it is in full development at the time of harvest, it has a short shelf life. In this work, it is showed that visible light and UV-C treatments not only delayed the senescence of broccoli, but also increased flavonoid content. Our results suggest that the most important enzyme in the phenylpropanoid biosynthesis pathway during broccoli postharvest is phenylalanine ammonia lyase, and that this may be a key point in regulating the biosynthesis of these nutritionally valuable compounds.

First Case of Glyphosate Resistance in Bromus catharticus Vahl.: Examination of Endowing Resistance Mechanisms.

Bromus catharticus Vahl. has been used as a valuable forage crop, but it has also been noted as a weed of winter crops and an invader in several countries. In Argentina, a putative glyphosate-resistant population of B. catharticus was identified as a consequence of the lack of effective control with glyphosate in the pre-sowing of wheat. Plant survival and shikimate accumulation analysis demonstrated a lower glyphosate-sensitivity of this population in comparison to a susceptible B. catharticus population. The resistant population was 4-fold more resistant to glyphosate than its susceptible counterpart. There was no evidence of target-site mechanisms of glyphosate resistance or an enhanced capacity to metabolize glyphosate in the resistant population. However, the resistant plants showed a lower foliar retention of glyphosate (138.34 µl solution g-1 dry weight vs. 390.79 µl solution g-1 dry weight), a reduced absorption of 14C-glyphosate (54.18 vs. 73.56%) and lower translocation of 14C-glyphosate from the labeled leaf (27.70 vs. 62.36%). As a result, susceptible plants accumulated a 4.1-fold higher concentration of 14C-glyphosate in the roots compared to resistant plants. The current work describes the first worldwide case of glyphosate resistance in B. catharticus. A reduced foliar retention of herbicide, a differential rate of glyphosate entry into leaves and an altered glyphosate translocation pattern would be the most likely mechanisms of glyphosate exclusion.

Expression of BoNOL and BoHCAR genes during postharvest senescence of broccoli heads.

BACKGROUND: Chlorophyll is the most abundant pigment on Earth, essential for the capture of light energy during photosynthesis. During senescence, chlorophyll degradation is highly regulated in order to diminish toxicity of the free chlorophyll molecule due to its photoactivity. The first step in the chlorophyll degradation pathway is the conversion of chlorophyll b to chlorophyll a by means of two consecutive reactions catalyzed by enzymes coded by NYC1 (NON-YELLOW COLORING 1), NOL (NYC1-LIKE) and HCAR. RESULTS: In this work, we studied the expression of NOL and HCAR genes during postharvest senescence of broccoli. We found that the expression of BoNOL increase during the first days of storage and then decrease. In the case of BoHCAR, its expression is maintained during the first days and then it also diminishes. Additionally, the effect of different postharvest treatments on the expression of these genes was also analyzed. It was observed that the expression of BoNOL is lower in the treatments performed with 1-methylcyclopropene (1-MCP), 6-benzylaminopurine (6-BAP) and modified atmospheres, while BoHCAR expression showed an increase in these same treatments, and a decrease in the treatment with ethylene. There were no variations in the expression of both genes in heat treatment, UV-C treatment and visible light treatment. CONCLUSIONS: These results suggest that both BoHCAR and BoNOL show a lower regulation of their expression than other genes involved in chlorophyll degradation during senescence. © 2020 Society of Chemical Industry.

Harvesting at different time-points of day affects glucosinolate metabolism during postharvest storage of broccoli.

The consumption of broccoli provides a large quantity of compounds with nutraceutical properties to the human diet. Broccoli has a high content of glucosinolates, compounds of the specialized metabolism with anticarcinogenic activity. In a previous work, we found that harvesting different time-points during the day affects the rate of senescence of broccoli heads during postharvest storage. In this work, we tested the same cultural practice to evaluate glucosinolate content and expression of genes involved in glucosinolate metabolism. Broccoli heads were harvested at 8:00, 13:00 and 18:00 h and stored for 5 d at 20 °C in darkness. We found that content and composition of the glucosinolate pool was affected by the time of harvest. Levels of indolic glucosinolates decreased with the time of harvest on the day whereas indolic glucosinolate showed only a moderate decrease. The expression of genes associated to the biosynthesis of aliphatic glucosinolates was variable during the day. In relation to indolic glucosinolates, an increase in the expression of the transcription factor BolMYB51 was detected around 13:00 h, which strongly correlated with the increase in expression of genes associated to their biosynthesis towards the end of the day. During postharvest, the storage in darkness affected differently the metabolisms of indolic and aliphatic glucosinolates. The content of aliphatics decreased during the postharvest period, as well as the expression of the genes associated with their biosynthesis. In contrast, in the case of indolics, their content remained constant or varied slightly, while the expression of the associated biosynthetic genes decreased only slightly. Finally, the genes related to the degradation of glucosinolates appeared to be strongly regulated by light conditions, since their expression increased during the course of the day and decreased markedly during postharvest storage in darkness. These results suggest that harvesting of broccolis close to noon would be convenient to maintain higher levels of glucosinolates during postharvest storage.

The time of the day to harvest affects the degreening, antioxidant compounds, and protein content during postharvest storage of broccoli.

Harvesting of broccoli at several moments of the day affects the rate of senescence during storage. In this work, broccoli heads were harvested at several moments and then kept at 20°C in order to analyze protein metabolism and antioxidant compounds. Almost no differences were detected in the contents of total and soluble proteins, and free amino acids. Only an increment in free amino acids was detected by day 3 in samples obtained at 8:00 hr. With reference to antioxidants, the contents of ascorbic acid, carotenoids and xanthophylls, phenols, and flavonoids were similar in samples harvested at different moments. However, an increment was detected in carotenoids, phenols, and flavonoids during storage of samples collected at 18:00 hr on day 3 and samples collected at 13:00 hr on day 5. The combination of delay of senescence and increment in antioxidants suggest harvesting at 12:00 or 18:00 hr. PRACTICAL APPLICATION: Broccoli is a vegetable with an important level of nutrients. However, it is also highly perishable and suffers a high rate of senescence and loss of quality during postharvest. In this work, it is demonstrated that the simple practice of harvests in different moments of the day can affect the postharvest behavior of broccoli, and it is suggested to carry out the harvest toward the end of the day.

Effects of ethylene, cytokinin and physical treatments on BoPaO gene expression of harvested broccoli.

BACKGROUND: Broccoli is a highly perishable vegetable that shows enhanced postharvest senescence and intense de-greening caused by chlorophyll degradation. One of the key steps of chlorophyll catabolism is the opening of chlorophyll tretrapyrrole catalysed by pheophorbide a oxygenase (PaO). In this study the expression of a gene encoding a putative PaO was characterised under several chemical and physical treatments. RESULTS: A fragment of a gene encoding a PaO from broccoli (BoPaO) was cloned. The expression of BoPaO showed an important increment during postharvest senescence, in correlation with chlorophyll degradation. Furthermore, broccoli heads were treated with the hormones cytokinin and ethylene. Cytokinin delayed the increment in BoPaO expression, while ethylene accelerated the process. Also, several postharvest treatments were applied in order to evaluate their effect on BoPaO expression. Samples treated with modified atmosphere, hot air, UV-C or white light showed a delay in chlorophyll degradation and de-greening. In most cases the treatments also delayed the increment in BoPaO expression during senescence. CONCLUSION: A close correlation between chlorophyll degradation and BoPaO expression was found during broccoli senescence. This relationship was corroborated in samples treated with different hormonal and physical applications.

Mitochondrial dysfunction affects chloroplast functions.

The transcriptomic response of A9:u-ATP9 and apetala3:u-ATP9 lines carrying a mitochondrial dysfunction in flower tissues has been characterized. Both lines showed an alteration in the transcription of several genes involved in carbon and nitrogen metabolism, stress responses, transcription factors and DNA binding proteins. Interestingly, several transcripts of photosynthetic-related genes were also affected in their expression such as the mRNAs encoding for chlorophyllase, chlorophyll binding proteins and a PSII. Moreover, chlorophyll levels were reduced and the Mg-dechelatase activity was increased, indicating an alteration in chlorophyll metabolism. Our results suggest that the mitochondrial dysfunction may also affect chloroplastic functions, and that our model could be useful to uncover retrograde signaling mechanisms operating between the three different plant genomes.

Effect of mitochondrial dysfunction on carbon metabolism and gene expression in flower tissues of Arabidopsis thaliana.

We characterized the transcriptomic response of transgenic plants carrying a mitochondrial dysfunction induced by the expression of the unedited form of the ATP synthase subunit 9. The u-ATP9 transgene driven by A9 and APETALA3 promoters induce mitochondrial dysfunction revealed by a decrease in both oxygen uptake and adenine nucleotides (ATP, ADP) levels without changes in the ATP/ADP ratio. Furthermore, we measured an increase in ROS accumulation and a decrease in glutathione and ascorbate levels with a concomitant oxidative stress response. The transcriptome analysis of young Arabidopsis flowers, validated by qRT-PCR and enzymatic or functional tests, showed dramatic changes in u-ATP9 plants. Both lines display a modification in the expression of various genes involved in carbon, lipid, and cell wall metabolism, suggesting that an important metabolic readjustment occurs in plants with a mitochondrial dysfunction. Interestingly, transcript levels involved in mitochondrial respiration, protein synthesis, and degradation are affected. Moreover, the levels of several mRNAs encoding for transcription factors and DNA binding proteins were also changed. Some of them are involved in stress and hormone responses, suggesting that several signaling pathways overlap. Indeed, the transcriptome data revealed that the mitochondrial dysfunction dramatically alters the expression of genes involved in signaling pathways, including those related to ethylene, absicic acid, and auxin signal transduction. Our data suggest that the mitochondrial dysfunction model used in this report may be useful to uncover the retrograde signaling mechanism between the nucleus and mitochondria in plant cells.

Effect of visible light treatments on postharvest senescence of broccoli (Brassica oleracea L.).

BACKGROUND: Broccoli (Brassica oleracea L.) is a rapidly perishable vegetable crop. Several postharvest treatments have been applied in order to delay de-greening. Since light has been shown to have an effect on pigment accumulation during development and darkness is known to induce senescence, the effect of continuous and periodic exposure to low-intensity white light at 22 °C on postharvest senescence of broccoli heads was assayed. RESULTS: Exposure to a constant dose of 12 micromol m(-2) s(-1) was selected as the most suitable treatment and was employed for subsequent experiments. During the course of the treatments, hue and L* values as well as chlorophyll content and visual observation of florets indicated an evident delay in yellowing in treated samples compared with controls. No statistically significant differences in total protein content were found, but soluble protein content was higher in treated samples. Total and reducing sugar as well as starch levels decreased during postharvest senescence, with lower values in control samples. CONCLUSION: The results of this study indicate that storage under continuous low-intensity light is an efficient and low-cost treatment that delays postharvest senescence while maintaining the quality of harvested broccoli florets.