Dissecting the biochemical and molecular-genetic regulation of diverse metabolic pathways governing aroma formation in indigenous aromatic indica rice varieties.

BACKGROUND: Aromatic rice is characterized by its distinct flavor and fragrance, imparted by more than 200 volatile organic compounds. The desirable trait of aroma relies on the type of the variety, with some varieties exhibiting considerably higher aroma content. This prompted us to undergo an exhaustive study of the aroma-associated biochemical pathways and expression of related genes, encoding the enzymes involved in those pathways in indigenous aromatic rice cultivars. METHODS AND RESULTS: The higher aroma level in aromatic rice varieties was attributed to higher transcript levels of PDH, P5CS, OAT, ODC, DAO and TPI, but lower P5CDH and BADH2, as revealed by comparative expression profiling of genes in 11 aromatic and four non-aromatic varieties. Some of the high-aroma containing varieties exhibited lower expression of SPDS and SPMS genes, concomitant with higher PAO expression. Protein immunoblot analyses showed lesser BADH2 protein accumulation in the aromatic varieties. The involvement of shikimate pathway in aroma formation was justified by higher levels of shikimic and ferulic acids due to higher expression of SK1, SK2 and ARD genes. The aromatic varieties exhibited higher expression of LOX3 and HPL, with higher corresponding enzyme activity, accompanied with lower accumulation of lipid hydroperoxides and higher level of total terpenoids, signifying the role of oxylipin pathway and terpene-related volatiles in aroma formation. The pattern of transcript level and metabolite accumulation followed the same trend in both vegetative (seedling) and reproductive (seed) tissues. Sequence analyses revealed several mutations in the upstream region and different exons and introns of BADH2 in the examined aromatic varieties. The allele specific marker system acted as fingerprint to distinguish the selected aromatic rice varieties. The cleaved amplified polymorphic sequence marker established the absence of any mutation in the 14th exon of BADH2 in the aromatic varieties. CONCLUSION: The present work clearly highlighted the biochemical and molecular-genetic mechanism of differential aroma levels which could be attributed to differential regulation of metabolites and genes, belonging to 2-acetyl-1-pyrroline, shikimate, oxylipin and terpenoid metabolic pathways in the indigenous aromatic rice varieties.

Fluoride tolerance in rice is negatively regulated by the 'stress-phytohormone' abscisic acid (ABA), but promoted by ABA-antagonist growth regulators, melatonin, and gibberellic acid.

The present manuscript aimed at investigating whether abscisic acid (ABA) promotes fluoride tolerance, similar to inciting salt adaptation in rice. Seeds of three salt-tolerant rice genotypes were maintained at 32 °C under 16/8 h light/dark photoperiodic cycle with 700 µmol photons m-2 s-1 intensity and 50% relative humidity in a plant growth chamber for 20 days. Suppressed ABA biosynthesis, and downregulated expression of ABA-inducible genes like Rab16A, Osem, and TRAB1 triggered NaCl-induced growth inhibition and physiological injuries like chlorophyll degradation, electrolyte leakage, formation of H2O2, malondialdehyde, and methylglyoxal in Matla. Reduced ABA accumulation increased the levels of melatonin and gibberellic acid in NaF (50 mg L-1)-stressed Nonabokra and Matla, which altogether promoted fluoride tolerance. Higher ABA content in NaF-stressed Jarava stimulated fluoride uptake via chloride channels, thus exhibiting severe fluoride susceptibility, in spite of higher production of ABA-associated osmolytes like proline, glycine-betaine and polyamines via the concerted action of genes like PDH, ADC, ODC, SAMDC, SPDS, SPMS, DAO, and PAO. Increased accumulation of compatible solutes in presence of high endogenous ABA promoted salt tolerance in Jarava; the same was insufficient to ameliorate fluoride-induced injuries in this cultivar. Treatment with ABA biosynthetic inhibitor, Na2WO4 promoted fluoride tolerance in Jarava, whereas further supplementation with exogenous ABA resulted in reversion back to fluoride-susceptible phenotype. Our work clearly established that ABA cannot always be considered as a 'universal' stress hormone as known in literature, since it acts as a negative regulator of fluoride tolerance which is more tightly regulated in rice via melatonin- and gibberellic acid-dependent pathways in ABA-independent manner.

Exogenous melatonin regulates endogenous phytohormone homeostasis and thiol-mediated detoxification in two indica rice cultivars under arsenic stress.

KEY MESSAGE: Melatonin enhanced arsenic (As) tolerance by inhibiting As bioaccumulation, modulating the expression of As transporters and phytohormone homeostasis, leading to efficient utilization of thiol machinery for sequestration and detoxification of this toxic metalloid. The present study was aimed at investigating the influence of exogenous melatonin on the regulation of endogenous plant growth regulators and their cumulative effects on metal(loid)-binding ligands in two contrasting indica rice cultivars, viz., Khitish (arsenic sensitive) and Muktashri (arsenic tolerant) under arsenic stress. Melatonin supplementation ameliorated arsenic-induced perturbations by triggering endogenous levels of gibberellic acid and melatonin, via up-regulating the expression of key biosynthetic genes like GA3ox, TDC, SNAT and ASMT. The endogenous abscisic acid content was also enhanced upon melatonin treatment by induced expression of the key anabolic gene, NCED3 and concomitant suppression of ABA8ox1. Enhanced melatonin content induced accumulation of higher polyamines (spermidine and spermine), together with up-regulation of SPDS and SPMS in Khitish, thereby modulating stress condition. On the contrary, melatonin escalated putrescine and spermidine levels in Muktashri, via enhanced expression of ADC and SAMDC. The role of melatonin appeared to be more prominent in Khitish, as evident from better utilization of thiol components like cysteine, GSH, non-protein thiols and phytochelatins, with higher GSH/GSSG ratio, despite down-regulated expression of corresponding thiol-metabolic genes (OsMT2 and OsPCS1) to deal with arsenic toxicity. The extent of arsenic bioaccumulation, which was magnified several folds, particularly in Khitish, was decreased upon melatonin application. Overall, our observation highlighted the fact that melatonin enhanced arsenic tolerance by inhibiting arsenic bioaccumulation, via modulating the expression levels of selected arsenic transporters (OsNramp1, OsPT2, OsPT8, OsLsi1) and controlling endogenous phytohormone homeostasis, leading to efficient utilization of thiol machinery for sequestration and detoxification of this toxic metalloid.

Exogenous supplementation of melatonin alters representative organic acids and enzymes of respiratory cycle as well as sugar metabolism during arsenic stress in two contrasting indica rice cultivars.

The objective of the present investigation was to understand the impact of exogenously applied melatonin on mitochondrial respiration and sugar metabolism in two contrasting rice cultivars, viz., Khitish (arsenic-susceptible) and Muktashri (arsenic-tolerant) under arsenic-stress. Melatonin effectively restored the level of organic acids like pyruvic acid, malic acid and more particularly citric acid by 33 % in Khitish which were lowered during arsenic-stress, whereas their levels were further elevated in Muktashri to provide energy for defence against arsenic-induced injury. Arsenic-exposure led to a significant inhibition in enzyme activities as well as corresponding transcript level of key respiratory enzymes, viz., pyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase, succinate dehydrogenase and malate dehydrogenase, intriguingly more prominently in case of Khitish. Conversely, melatonin supplementation, irrespective of cultivars, considerably improved the activity of the above enzymes and corresponding gene expressions during stress, indicating acceleration in the rate of Krebs cycle. Melatonin supplementation also stimulated the accumulation of total soluble sugars by 62 % and 25 %, reducing sugars by 50 % and 44 % and non-reducing sugars by 75 % and 14 % in Khitish and Muktashri respectively, concomitant with higher activities of acid invertase, sucrose synthase and sucrose phosphate synthase enzymes, along with the expression of corresponding genes. Enhanced starch accumulation via regulation of alpha amylase and starch phosphorylase activities and gene expression, by melatonin also contributed towards better stress tolerance. Overall, this work illustrated the efficacy of melatonin in the regulation of representative organic acids and enzymes of respiratory cycle along with starch and sugar metabolism in rice cultivars under arsenic toxicity.

Silicon-regulated antioxidant and osmolyte defense and methylglyoxal detoxification functions co-ordinately in attenuating fluoride toxicity and conferring protection to rice seedlings.

The objective of this manuscript was to demonstrate the efficacy of silicon supplementation in relieving the fluoride-induced damages in rice cultivar, Khitish. The exposure of seedlings to two different concentrations of fluoride, viz., 25 and 50 mg L-1 NaF caused increase in fluoride accumulation, as a result of which the seedlings suffered severe oxidative stress, as evident from growth inhibition, reduction in seed germination, tissue biomass, root and shoot length, decline in chlorophyll content, increases in electrolyte leakage, H2O2 content, lipid peroxidation (malondialdehyde content and lipoxygenase activity), protein carbonylation and protease activity. The extent of damage was more at higher fluoride concentration. Silicon amendment, irrespective of fluoride concentrations, led to large build-up of endogenous silicon level and brought considerable improvement in all the parameters examined with respect to fluoride stress. The fluoride-mediated enhancement in methylglyoxal level was lowered by silicon, because of the prominent activation of glyoxalase I and glyoxalase II. While the stress-mediated induction in antioxidative enzymes like GPOX, APX, SOD, GPX and GR was lowered by silicon, the inhibition in CAT activity was relieved. The antioxidative defense mechanism was also boosted up via enhanced content of total phenolics and carotenoids. However, the fluoride-mediated increase in anthocyanins, flavonoids, xanthophyll, ascorbate and reduced glutathione, and osmolytes like total amino acids, proline and glycine-betaine, were all lowered in presence of silicon, together with reduced PAL and P5CS activity. Overall, silicon reduced oxidative damages to develop fluoride-tolerant rice plants through augmentation of different antioxidant and osmolyte defense and methylglyoxal detoxification system.

Spermine ameliorates prolonged fluoride toxicity in soil-grown rice seedlings by activating the antioxidant machinery and glyoxalase system.

The current manuscript presents the first report on the ameliorative roles of exogenous spermine (Spm) during prolonged fluoride-induced toxicity and oxidative damages in the susceptible rice cultivar, IR-64. The application of Spm increased the overall growth in the stressed seedlings by significantly restricting fluoride bioaccumulation within the shoots and roots. The Spm-treated stressed seedlings exhibited low chlorosis and induced activity of pyruvate dehydrogenase and nitrate reductase due to reduced accumulation and localization of reactive oxygen species (ROS) in the shoot and root. Spm-supplementation during stress reduced the levels of molecular damages by lowering malondialdehyde, electrolyte leakage and protein carbonylation, and lipoxygenase and protease activity due to effective detoxification of ROS by the antioxidants like proline, glycine-betaine, anthocyanin, flavonoids, phenolics and higher polyamines like Spm and spermidine. Excessive accumulation of the toxic methylglyoxal was reversed due to the activation of the glyoxalase system (comprising of glyoxalase I and II) and the ascorbate-glutathione cycle. Exogenous Spm also triggered the activity of superoxide dismutase, guaiacol peroxidase, glutathione peroxidase and phenylalanine ammonia lyase, which efficiently scavenged ROS in the stressed seedlings. Overall, Spm treatment mitigated the fluoride-induced injuries in IR-64 by reducing fluoride bioaccumulation and elaborately refining the various defence machineries.

Spermidine application reduces fluoride uptake and ameliorates physiological injuries in a susceptible rice cultivar by activating diverse regulators of the defense machinery.

The manuscript illustrates the ameliorative effects of exogenously applied higher polyamine (PA), spermidine (Spd) in the susceptible indica rice cultivar IR-64 subjected to prolonged fluoride stress. The Spd treatment drastically reduced fluoride bioaccumulation by restricting entry of the anions through chloride channels and enabled better maintenance of the proton gradient via accumulation of P-H+/ATPase, thereby improving the root and shoot lengths, fresh and dry weights, RWC, chlorophyll content and activities of pyruvate dehydrogenase (PyrDH), α-amylase, and nitrate reductase (NR) in the Spd-treated, stressed plants. Expression of RuBisCo, PyrDH, α-amylase, and NR was stimulated. Spd supplementation reduced the molecular damage indices like malondialdehyde, lipoxygenase, protease activity, electrolyte leakage, protein carbonylation, H2O2, and methylglyoxal (detoxified by glyoxalase II). Mitigation of oxidative damage was facilitated by the accumulation and utilization of proline, glycine-betaine, total amino acids, higher PAs, anthocyanin, flavonoids, ß-carotene, xanthophyll, and phenolics as verified from the expression of genes like P5CS, BADH1, SAMDC, SPDS, SPMS, DAO, PAO, and PAL. Spd treatment activated the ascorbate-glutathione cycle in the stressed seedlings. Expression and activities of enzymatic antioxidants showed that GPOX, APX, GPX, and GST were the chief ROS scavengers. Exogenous Spd promoted ABA accumulation by upregulating NCED3 and suppressing ABA8ox1 expression. ABA-dependent osmotic stress-responsive genes like Osem, WRKY71, and TRAB1 as well as ABA-independent transcription factor encoding gene DREB2A were induced by Spd. Thus, Spd treatment ameliorated fluoride-mediated injuries in IR-64 by restricting fluoride uptake, refining the defense machinery and activating the ABA-dependent as well as ABA-independent stress-responsive genes.

Carbohydrate content and antioxidative potential of the seed of three edible indica rice (Oryza sativa L.) cultivars.

Rice (Oryza sativa L.) grains or seeds are known to lose much of their nutrient and antioxidant contents, following polishing. The current study was undertaken to evaluate and compare the carbohydrate content and antioxidant parameters in the unpolished and polished seeds of three edible indica rice cultivars, namely Swarna (SW), the most popular indica rice cultivar in India and aromatic or scented cultivars Gobindobhog (GB) and Pusa Basmati (PB). While both the sucrose and starch content was the maximum in PB seeds (both unpolished and polished), the amylose content was the highest in SW polished seeds. SW polished seeds were superior as compared to GB and PB cultivars in terms of total antioxidant capacity, DPPH radical scavenging and Fe(II) chelation potential, as well as the highest lipoxygenase (LOX) inhibition or H2O2 scavenging potential, probably due to the maximum accumulation of total phenolics and flavonoids, the two important antioxidants. The reducing power ability was, however, identical in both SW and GB polished seeds. The PB polished seeds were more potent in superoxide and hydroxyl scavenging, whereas GB in nitric oxide (NO) scavenging. The common observation noted after polishing of seeds was the reduction in the level of carbohydrates and antioxidant potential, though the extent of reduction varied in the three cultivars. The only exception was GB, where there was no alteration in NO scavenging potential even after polishing. Our study showed the better performance of SW polished seeds with respect to higher amylose content and majority of the tested parameters governing antioxidant capacity and radical scavenging potential, thus highlighting the greater dietary significance of SW over the other two cultivars.