Yong-Hua Yang

Nitric oxide regulates shikonin formation in suspension-cultured Onosma paniculatum cells.

Endogenously occurring nitric oxide (NO) is involved in the regulation of shikonin formation in Onosma paniculatum cells. NO generated after cells were inoculated into shikonin production medium reached the highest level after 2 d of culture, which was 16 times that at the beginning of the experiment, and maintained a high level for 6 d. A nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine (L-NNA), and a nitrate reductase (NR) inhibitor, sodium azide (SoA), consistent with their inhibition of NO biosynthesis, decreased shikonin formation significantly. This reduction could be alleviated or even abolished by exogenous NO supplied by sodium nitroprusside (SNP), suggesting that the inhibition of NO biosynthesis resulted in decreased shikonin formation. However, when endogenous NO biosynthesis was up-regulated by the elicitor from Rhizoctonia cerealis, shikonin production was enhanced further, showing a dependence on the elicitor-induced NO burst. Real-time PCR analysis showed that NO could significantly up-regulate the expression of PAL, PGT and HMGR, which encode key enzymes involved in shikonin biosynthesis. These results demonstrated that NO plays a critical role in shikonin formation in O. paniculatum cells.

Brassinolide Amelioration of Aluminum Toxicity in Mungbean Seedling Growth

Abstract Brassinolide (BL) is known to be essential for plant growth and development and regarded as a new class of plant hormones. The ameliorative effects of BL‐treatment on stressed plants have been reported. However, little is known about the effects of BL on plant growth under aluminum (Al) stress. In the present study, mungbean (Phaseolus aureus Roxb.) seedlings were grown in a growth chamber in solution with combined concentrations of Al (0, 2, and 5 mM) and BL (0, 0.1, 10, 1000, 100,000 ng L−1) under a randomized complete block design experiment for 2 weeks, to evaluate the ameliorative effects of BL on Al toxicity for the seedling growth under Al stress. Results showed that BL had promoted growth of mungbean seedlings under Al stress. In the absence of Al stress, BL had no significant effect in increasing epicotyl length of mungbean seedlings, while in the presence of Al stress, epicotyl length showed significant increase under Al stress in the presence of BL. A similar result was obtained in seedling height and root length under 2 and 5 mM Al stress. Brassinolide also significantly increased fresh weights of shoots and roots under Al stress. Interestingly, BL at 0.1 ng L−1 had significant differential response in dry weight distributed between shoots and roots under Al stress, with no effect in dry weight of shoots, but significant increase in root dry weight. Brassinolide also significantly increased chlorophyll content under Al stress. However, the most significant promotive effect of BL on morphological traits and chlorophyll content seems to depend on the addressed traits, BL concentration, and Al stress. The mechanisms for differential response in dry weight between shoots and roots by BL at low concentration and increase of chlorophyll content under Al stress needs to be addressed further.

Boron amelioration of aluminum toxicity in mungbean seedlings

Abstract Boron (B) amelioration of aluminum (Al) toxicity was studied for growth of mungbean (Phaseolus aureus Roxb.) seedlings and cuttings (without roots) in a growth chamber. Mungbean seedlings and cuttings were grown in the solution with combinations of three concentrations of B (0,5, and 50 μM) and three concentrations of Al (0, 2, and 5 mM) in randomized complete block design experiments for 16 days. Results showed that B significantly promoted elongation of epicotyls and hypocotyls, and increased height of seedlings grown under Al stress. Boron also increased fresh weight of seedlings in high Al solution. Treatment of plants grown with high B and Al stress had no apparent effect on fresh and dry weights of seedling roots. Seedling dry weight increased significantly by adding high B to solutions with 2 mM or 5 mM Al. No significant differences were observed between the high B treatment and the control (normal B, 5 μM) in lengths of epicotyls and fresh and dry weights of mungbean cuttings grown under...

Expression analysis of shikonin-biosynthetic genes in response to M9 medium and light in Lithospermum erythrorhizon cell cultures

When cells of Lithospermum erythrorhizon were transferred from B5 solid medium into M9 liquid medium and grown in the dark, they produced and accumulated shikonin and its derivatives; meanwhile, transcripts of PAL, 4CL, HMGR, PGT, and CYP98A6 were rapidly induced within 2xa0h, peaking within 6xa0h, and then decreasing over time. However, when L. erythrorhizon cells were cultured in M9 medium and grown under white light, a negative regulator for the biosynthesis of shikonin and its derivatives, the “stimulating effect” of the medium transition on the transcription of these genes was also displayed with similar patterns over 24xa0h of culture period. To investigate the time-dependent effect of light signals on regulating gene transcription, expression analysis of L. erythrorhizon dark-inducible genes LeDI-1–LeDI-5 was conducted. No obvious inhibitory effects of light signals on the transcription of LeDI-1–LeDI-5 were detected during the first 12xa0h, but these genes, in particular LeDI-2 and LeDI-4, were suppressed by light signals to some degree during longer culture periods, 12xa0h to 10xa0days. These findings suggest that light signals are likely to inhibit the transcription of the shikonin-biosynthetic genes indirectly or under long-term conditions.

Differential Responses of Anti-Oxidative Enzymes to Aluminum Stress in Tolerant and Sensitive Soybean Genotypes

ABSTRACT Seedlings of two soybean genotypes, BX10 [aluminum (Al)-tolerant] and BD2 (Al-sensitive), were treated with Al to evaluate the relative root growth (RRG), callose content, Al-sensitive zone, lipid peroxidation, and the anti-oxidative enzyme activities by histochemical and biochemical assays. Under Al toxicity, the RRG reduction of BD2 was more significant than that of BX10, while callose content displayed a contrary trend. The 2–5 mm zone of root apex was the main Al-sensitive zone for soybeans. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were found to be higher in the two genotypes under Al toxicity than that of the controls. The SOD, POD, and CAT activities of BX10; however, were significantly lower than those of BD2. These results implied that producing low quantity of ROS may be one aspect of the Al-tolerant mechanism for soybeans, which in turn helps them adapt to Al stress.

Silicon effects on aluminum toxicity to mungbean seedling growth

Abstract The effects of silicon (Si) on the toxicity of aluminum (Al) to mungbean (Phaseolus aureus Roxb.) seedlings were studied in a growth chamber. Mungbean seedlings were grown in a nutrient solution with combinations of three concentrations of Si (0,1, and 10 mM) and three concentrations of Al (0, 2, and 5 mM) in randomized completely block design experiments for 16 days. Silicon at 1 mM in the solution decreased root length, fresh and dry weights, and chlorophyll content, and showed no significant effect on epicotyl length and seedling height, and protein contents of shoots or roots in mungbean seedling under no Al stress. But, Si at 10 mM showed marked toxic effects on mungbean seedling growth and increased protein contents of the shoots or roots. In contrast, under 2 mM Al stress, Si addition at 1 mM had significant increasing effect on root length, fresh and dry weights, and chlorophyll content. It also had decreasing effect on protein contents of the shoots or roots, and had no effect on epicoty...

Effect of aluminium and 6‐benzylaminopurine on the growth of the epicotyl in mungbean seedlings

Abstbact Aluminium (Al) toxicity was studied for mungbean (Phaseolus aureus Roxb.) seedlings under controlled environmental conditions in a growth chamber. Ten‐day‐old seedlings or cuttings (root removed) grew in dilute nutrient solution (pH 4.1) with Al levels and 6‐benzylaminopurine (6‐BA) concentrations for six days. Results showed that Al concentrations less than 2 mM had no significant effect on the length of the epicotyl in mungbean seedlings. Elongation of the epicotyl of intact seedlings was inhibited by 5 mM Al. The decrease in dry weight of the epicotyl was more evident than that in dry weight of hypocotyl, root, and primary leaves, indicating that epicotyl damage was greater than other organs by Al toxicity. No significant difference was found in the length of epicotyl between seedlings with and without 6–3A treatment. But there was a marked decrease in length of the epicotyl when cuttings were treated with 1 mM and 5 mM Al. Addition of 6‐BA to the nutrient solution significantly increased the ...

EFFECTS OF METHYL JASMONATE WITH INDOLE-3-ACETIC ACID AND 6-BENZYLAMINOPURINE ON THE SECONDARY METABOLISM OF CULTURED ONOSMA PANICULATUM CELLS

SummaryMethyl jasmonate (MeJA) interacted significantly with both indole-3-acetic acid (IAA) and 6-benzylaminopurine (BA) to influence cell growth of cultured Onosma paniculatum cells. Cell growth decreased with increasing concentrations of MeJA from 0.004–4.45 μM with or without IAA and BA. The same concentrations of MeJA (0–4.45 μM) increased the cell growth with IAA and BA, when administered to the cultured cells in M9 medium. This was found to enhance the production of shikonin. The optimum time for MeJA addition for enhanced shikonin formation was 4 d after cell inoculation in M9 medium. Furthermore, shikonin formation was affected significantly by both MeJA/IAA and MeJA/BA combinations. Shikonin content was enhanced by increasing MeJA concentrations with IAA concentrations in the range of 0–28 μM and with BA concentrations in the range of 0–44.38 μM in MeJA/BA experiments, respectively. The optimal combination of MeJA and IAA was 4.45 μM and 0.28 μM, while MeJA and BA concentrations of 4.45 μM and 2.22 μM were optimal for shikonin formation. The result also showed that MeJA increased phenylalanine ammonia-lyase (PAL) and p-hydroxybenzoic acid-geranyltransferase (PHB-geranyltransferase) activites during the course of shikonin formation, but decreased the activity of PHB-O-glucosyltransferase within 9 d after inoculation. These results suggest that enhanced shikonin formation in cultured Onosma paniculatum cells induced by MeJA involves regulation of the key enzyme activities.

Physiological effects of aluminum/calcium ratios on aluminum toxicity of mungbean seedling growth

Mungbean seedlings were grown in a nutrient solution with combined concentrations of aluminum (Al) (0, 2, and 5 mM) and calcium (Ca) (0–10 mM) in a randomized complete block design experiment for 16 days. The results showed that under no Al stress, Al0 + Ca0 decreased chlorophyll content by 10%, increased soluble protein, free proline content, and electrolyte leakage by 51%, 533%, and 111%, respectively, compared with a control (Al0 + Ca0.5). Under 2mMAl stress, Al2 + Ca0 decreased chlorophyll content by 19%, increased soluble protein, free proline content, and electrolyte leakage by 48%, 405%, and 55%, respectively, compared with a control (Al2 + Ca0.5). The ratio of Al/Ca = 1:2 increased chlorophyll content by 3%, decreased free proline content and electrolyte leakage by 64% and 34%, respectively. However, all of Al/Ca = 2:1, 1:1 and 1:2 at 2AlmM significantly increased soluble protein, compared with Al2 + Ca0.5. Under 5mMAl stress, Al5 + Ca0 decreased chlorophyll content by 11%, and increased soluble protein, free proline content, and electrolyte leakage by 77%, 85%, and 30%, respectively, compared with a control (Al5 + Ca0.5). The ratio of Al/Ca = 1:2 increased chlorophyll content by 14%, decreased free proline content by 80%, and electrolyte leakage by 34%, compared with Al5 + Ca0.5. However, all ratios of Al/Ca = 2:1, 1:1 and 1:2 at 5mMAl also significantly increased soluble protein, compared with the control. Furthermore, it was observed that Al/Ca = 1:2 could accumulate more Al and Ca in the seedlings than Al/Ca = 2:1 and 1:1 regardless of Al stress (2mM or 5mMAl). Lack of Ca affected the synthesis of chlorophyll, increased soluble protein and free proline, and enhanced electrolyte leakage. Al/Ca = l:2 might be an appropriate ratio for physiological alleviation of Al toxicity to mungbean seedling growth. High concentration of Ca in the solution at appropriate Al/Ca ratio (e.g. Al/Ca = 1:2) could increase chlorophyll content, an indication of alleviation effect. On the contrary, the high Ca caused a significant increase of soluble protein. It suggests the involvement of various proteins that have properties to resist Al toxicity for mungbean seedling growth.

Effect of citric acid on aluminum toxicity in the growth of mungbean seedlings

Abstract Citric acid was reported to chelate aluminum (Al) strongly and alleviate Al toxicity. Detoxification of Al by adding citric acid was studied in experiments with mungbean (Phaseolus aureus Roxb.) seedlings and cuttings (without roots) grown in a growth chamber. The seedlings and cuttings were grown for sixteen days in a dilute nutrient (pH 4.1) at two concentrations of citric acid (0, 5 mM) and three concentrations of Al (0, 2, and 5 mM) using a complete randomized design method. Results showed that 5 mM citric acid increased the height of mungbean seedlings, length of epicotyl, and fresh weight of seedlings and roots significantly. Although the mitigation by adding citric acid was more effective at higher Al concentrations, it seemed that citric acid was harmful to fresh and dry weights of cuttings under higher Al stress. This was perhaps attributed to the absence of roots. The content of soluble protein and chlorophyll in the seedlings were decreased under high Al stress by adding citric acid, b...