Stress responses in two genotypes of mulberry (Morus alba L.) under NaCl salinity

Changes in biomass yield rates, cell membrane stability (CMS), malondialdehyde (MDA) content and in the levels of physiological stress markers such as proline and glycine betaine in two high yielding genotypes (S1 and ATP, salt tolerant and salt sensitive, respectively) of mulberry under NaCl salinity were studied. Biomass yield rates and CMS were significantly decreased in both the genotypes under stress conditions. Per cent of decrease in biomass yield rate and CMS was relatively less in S1 than in ATP. Salt stress results a significant increase in the accumulation of proline, by 6-fold in S1 and 4-fold in ATP. Glycine betaine content was also increased significantly in stressed plants. However, the per cent increase was more in S1 than in ATP. The level of lipid peroxidation as indicated by MDA formation was greater in ATP than in S1. These results clearly support the better salt tolerant nature of S1 compared to ATP genotype.     

Agrobacterium-mediated transient MaFT expression in mulberry (Morus alba L.) leaves

To optimize Agrobacterium-mediated transient transformation assay in mulberry (Morus alba L.), various infiltration methods, Agrobacterium tumefaciens (A. tumefaciens) strains, and bacterial concentrations were tested in mulberry seedlings. Compared with LBA4404, GV3101 harboring pBE2133 plasmids presented stronger GUS signals at 3 days post infiltration using syringe. Recombinant plasmids pBE2133:GFP and pBE2133:GFP:MaFT were successfully constructed. Transient expression of MaFT:GFP protein was found in leaves, petiole (cross section), and shoot apical meristem (SAM) of mulberry according to the GFP signal. Moreover, MaFT:GFP mRNA was also detected in leaves and SAM via RT-PCR and qRT-PCR. An efficient transient transformation system could be achieved in mulberry seedlings by syringe using A. tumefaciens GV3101 at the OD₆₀₀ of 0.5. The movement of MaFT expression from leaves to SAM might trigger the precocious flowering of mulberry.     

Comparative proteomic analysis provides new insights into mulberry dwarf responses in mulberry (Morus alba L.)

Mulberry dwarf (MD) is a serious infectious disease of mulberry caused by phytoplasma. Infection with MD phytoplasma results in stress phenotypes of yellowing, phyllody, stunting, proliferation, and witches' broom. Physiological and biochemical analysis has shown that infection with MD phytoplasma causes an increase in soluble carbohydrate and starch content, and a decrease in the net photosynthesis rate, carboxylation efficiency, and pigment content of leaves. Furthermore, damage to the chloroplast ultrastructure was detected in infected leaves. To better understand the pathogen‐stress response of mulberry (Morus alba L.) to MD phytoplasma, we conducted a comparative proteomic analysis using 2‐DE of infected and healthy leaves. Among 500 protein spots that were reproducibly detected, 20 were down‐regulated and 17 were up‐regulated. MS identified 16 differentially expressed proteins. The photosynthetic proteins rubisco large subunit, rubisco activase, and sedoheptulose‐1,7‐bisphosphatase showed enhanced degradation in infected leaves. Based these results, a model for the occurrence mechanism of MD is proposed. In conclusion, this study provides new insights into the mulberry response to MD phytoplasma infection.     

Modulations in key enzymes of nitrogen metabolism in two high yielding genotypes of mulberry (Morus alba L.) with differential sensitivity to salt stress

Effect of salinity stress on the performance of nitrogen metabolism was studied in two high yielding genotypes of mulberry with differential sensitivity to NaCl (S1 and ATP, salt tolerant and susceptible, respectively). Three-month-old healthy mulberry plants were subjected to different regimes of NaCl stress [0.0 (control), 0.5, 1.0 and 1.5% NaCl] and leaf samples were collected on 4, 8 and 12 DAT (days after treatment) for the analysis. The activities of nitrate reductase (NR: EC 1.6.6.1), nitrite reductase (NiR: EC 1.6.6.4), protease, glutamine synthetase (GS: EC 6.3.1.2) and its accumulation pattern, glutamate synthase (GOGAT: EC 1.4.1.13), glutamate dehydrogenase (NADH-GDH: EC 1.4.1.2 and NADPH-GDH: EC 1.4.1.4), aspartate aminotransferase (AAT: EC 2.6.1.1) and alanine aminotransferase (ALAT: EC 2.6.1.2) coupled with total protein content, free amino acid level and ammonia content were studied in leaves of both genotypes of mulberry. The total protein content in leaves of both genotypes declined with progressive accumulation of free amino acid levels. Further, the decrease in protein content was less in S1 than ATP, and it was correlated with protease activity, ammonia content and accumulation of free amino acid levels. Higher free amino acid levels were registered for S1 than ATP at 1.0 and 1.5% NaCl stress and on all days of sampling. Ammonia content was increased in both genotypes and comparatively higher ammonia levels were recorded for ATP. Increased NaCl concentrations lead to a decrease in the activity of NR and NiR in both the genotypes, the decrease was more pronounced in ATP than S1. The enhanced activity of GDH (NADH and NADPH) was noticed in both genotypes, whereas the NADPH-GDH activity was found relatively higher in S1. The immunoblot analysis with GS-45 antibodies revealed a specific cross-reaction with 42 and 45 kDa proteins in S1, and only 45 kDa protein in ATP genotype. However, increased GS protein accumulation pattern (both 42 and 45 kDa) was observed in S1 under high NaCl. Whereas, accumulation of 45 kDa protein was unchanged at all levels of stress and slight accumulation in 42 kDa protein at 1.5% NaCl was observed for ATP. Elevation in the enzyme activities of GS, GOGAT were coupled with AAT and ALAT observed in both the genotypes. Higher enzymatic activities of S1 than ATP under salinity stress may be due to efficient capacity of ammonia detoxification. Salt tolerance of S1 supports the higher metabolic activity under salinity leading to lesser amount of ammonia accumulation and higher levels of free amino acid in the tissue. In agreement with these results the physiological significance of enzymatic changes and ammonia assimilation during salt stress in relevance to plant nitrogen metabolism was discussed.     

In vitro induction of tetraploidy in mulberry (Morus alba L.)

A high frequency of tetraploidy was induced in mulberry (Morus alba L.) through apical bud treatment under in vitro conditions. Apical buds from in vitro-grown plants were treated with three different concentrations (0.05, 0.1 and 0.2%) of colchicine in MS medium for 24 h. Tetraploidy at a frequency of 39.4±4.8% was obtained using 0.1% colchicine, whereas the frequency of tetraploidy was significantly reduced to 16.7±2.3% when 0.2% colchicine was used. Morphological, histological and cytological evidence indicated a phenotypic and genomic similarity of in vitro- with ex vitro-induced tetraploids. Rooting of tetraploids was on basal medium containing 2.6 μm NAA. The recovery of tetraploids was 80.8% more efficient using the in vitro method instead of the ex vitro method. The use of the same colchicine medium for up to 4 weeks with additional explants was found to be equally effective for the induction of tetraploidy. Received: 6 January 1997 / Revision received: 6 October 1997 / Accepted: 12 December 1997     

Oxidative stress and antioxidant responses of mulberry (Morus alba) plants subjected to deficiency and excess of manganese

The aim of the study was to relate the effects of deficiency and excess of Mn with the generation of reactive oxygen species (ROS) and altered cellular redox environment in mulberry (Morus alba L.) cv. Kanva-2 plants. Mn deficiency symptom appeared as mild interveinal chlorosis in middle leaves. Mn-excess did not produce any specific symptom. Leaf water potential (Ψ) was increased in Mn-deficient and Mn-excess mulberry plants. Mn-deficient leaves contained less Mn, less chloroplastic pigments and high tissue Fe, Zn and Cu concentrations. Starch content was increased with increasing Mn supply. While reducing sugar content increased in Mn-deficient and Mn-excess plants as well, non-reducing sugars remained unaffected in Mn-deficient plants and decreased in Mn-excess plants. Moreover, study of antioxidative responses, oxidative stress (H₂O₂ and lipid peroxidation) and cellular redox environment [dehydroascorbate (DHA)/ascorbic acid (AsA) ratio] in Mn-stressed mulberry plants was also undertaken. Both hydrogen peroxide and lipid peroxidation were enhanced in the leaves of Mn-deficient plants. Increased H₂O₂ concentration in Mn-excess leaves did not induce oxidative damage as indicated by no change in lipid peroxidation. The ratio of the redox couple (DHA/AsA) was increased both in Mn-deficient or Mn-excess plants. The activities of superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) increased in Mn-deficient plants. The activity of ascorbate peroxidase (EC 1.11.1.11) increased with increasing Mn supply. The results suggest that deficiency or excess of Mn induces oxidative stress through enhanced ROS generation and disturbed redox couple in mulberry plants.     

Mulberry leaves (Morus alba L.) ameliorate obesity-induced hepatic lipogenesis,fibrosis, and oxidative stress in high-fat diet-fed mice

Obesity is associated with chronic diseases such as fatty liver, type 2 diabetes, cardiovascular disease, and severe metabolic syndrome. Obesity causes metabolic impairment including excessive lipid accumulation and fibrosis in the hepatic tissue as well as the increase in oxidative stress. In order to investigate the effect of mulberry leaf (Morus alba L.) extract (MLE) on obesity-induced oxidative stress, lipogenesis, and fibrosis in liver, MLE has been gavaged for 12 weeks in high-fat diet (HFD)-induced obese mice. MLE treatment significantly ameliorated LXRα-mediated lipogenesis and hepatic fibrosis markers such as α-smooth muscle actin, while MLE up-regulated lipolysis-associated markers such as lipoprotein lipase in the HFD-fed mice. Moreover, MLE normalized the activities of antioxidant enzymes including heme oxygenase-1 and glutathione peroxidase in accordance with protein levels of 4-hydroxynonenal in the HFD-fed mice. MLE has beneficial effects on obesity-related fatty liver disease by regulation of hepatic lipid metabolism, fibrosis, and antioxidant defense system. MLE supplementation might be a potential therapeutic approach for obesity-related disease including non-alcoholic fatty liver disease.     

Changes in nitrogen levels and free amino acids in rooting cuttings of mulberry (Moms alba)

Total and protein nitrogen in bark and wood of parent stems of mulberry ( Morns alba L. cv. Ichinose) decreased readily and to the same extent during leafing-out of the buds, but the decrease in wood was less marked than in bark. Simultaneously, soluble nitrogen in both bark and wood also declined but the depletion was less marked than that of total and protein nitrogen. During the same period total nitrogen in the new shoots and adventitious roots increased drastically; however, the increase in total nitrogen in the growing parts during rooting was almost the same as the decrease in total nitrogen in the parent stems. Proline, the prevalent amino acid in wood and bark of the parent stems, decreased drastically during rooting, whereas during the same period asparagine in the developing buds, callus and adventitious roots increased markedly and became the predominant amino acid. The amount of arginine was relatively high in bark of the parent stems but Low in wood and the buds. The level of arginine in bark decreased considerably during the experiments (as did that of proline). The results suggest that the nitrogen required by the growing parts (sinks) in the rooting cuttings comes mainly from protein breakdown in bark of the parent stems (source), although stored protein in wood (source) and soluble nitrogen in bark and wood (sources) also play a part in storage of nitrogen. Asparagine is suggested to be the main nitrogen transport compound in the new growth of the tree and the initiating roots of cuttings.     

Regeneration of plants from the culture of leaves and axillary buds in mulberry (Morus indica L.)

Stem segments, axillary buds and leaves excised from established shoot cultures of Morus indica were soaked in MS liquid medium containing benzyladenine (0.5, 1, 2 mg/1) and were cultured subsequently on semi solid medium of the same composition. Numerous shoot buds differentiated from leaf and axillary buds but stem segments were unresponsive. The shoot buds on isolation and culture developed into plantlets. Callus tissues which developed at the base of the leaf explant upon subculture also differentiated numerous shoot buds.Abbreviations BA benzyl adenine - CM coconut milk - 2, 4-D 2, 4 dichlorophenoxy acetic acid - Kn kinetin - MS Murashige and Skoog - Z zeatin     

Control of hyperglycemia and retardation of cataract by mulberry (Morus indica L.) leaves in streptozotocin diabetic rats

Dried leaf powder of mulberry (M. indica L.) when given along with the diet at 25% level to streptozotocin induced diabetic male Wistar albino rats for 8 weeks, controlled hyperglycemia, glycosuria, albuminuria and retarded onset of retinopathy. Untreated diabetic rats showed hyperglycemia, glycosuria, albuminuria and developed lenticular opacity after 8 weeks of experimental period.     

Chemical characterization of cytotoxic indole acetic acid derivative from mulberry fruit (Morus alba L.) against human cervical cancer

The fruit of the white mulberry tree (Morus alba L.) is a multiple fruit with a sweet flavor commonly consumed around the world. Chemical investigation of the fruits led to the isolation of two indole acetic acid derivatives (1 −2) including a new compound, which turned out to be an isolation artifact, 3S-(β-D-glucopyranosyloxy)-2,3-dihydro-2-oxo-1H-indole-3-acetic acid butyl ester (1), along with five known compounds (3 −7). Compounds 2 and 7 were newly identified from mulberry fruit. The new isolation artifact (1) exhibited cytotoxic effect on human cervical cancer Hela cells in a dose-dependent manner. Compound 1 activated caspase-8, caspase-9, and caspase-3, followed by cleavage of PARP, a substrate of caspase-3, in a dose-dependent manner. Simultaneous alterations in protein expression of mitochondrial factors Bax, BID and Bcl-2 were also observed. A comparison between compounds 1 and 2 led to a structure-activity relationship analysis of the cytotoxic effect. These results suggest that compound 1 could be beneficial in human cervical cancer treatment, and provide a theoretical basis for further application of compound 1.     

Phytostilbenoid production in white mulberry (Morus alba L.) cell culture using bioreactors and simple deglycosylation by endogenous enzymatic hydrolysis

Phytostilbenes are responsible for several biological activities of mulberry (Morus sp.), which has been widely used as a raw material in health products. This study aimed to investigate the capability of Morus alba L. cell in bioreactors to produce the major bioactive stilbenes. The cell obtained from air-driven bioreactors such as round bottom, flat bottom, and air-lift vessel shape bioreactors was collected and analyzed for the levels of mulberroside A and oxyresveratrol. The results showed that the cell culture in round bottom and air-lift vessel bioreactors had higher growth rate, as compared with the cell culture in shake flasks (1.38- and 1.41-fold, respectively). The optimized culture condition to produce mulberroside A was obtained from round bottom bioreactor culture (55.56 ± 11.41 μmol/L). Additionally, endogenous stilbenoid hydrolysis of cell from the bioreactor culture was examined. Under optimized hydrolytic conditions, mulberroside A in the cell was readily deglycosylated to give oxyresveratrol within 1 h. These results indicated that the glycoside mulberroside A in the cell is sensitive to the endogenous enzymatic hydrolysis. Interaction of the stilbenoid components with the endogenous hydrolytic enzyme triggered by cell disruption in M. alba samples was suggested to be the major cause of the alteration of the stilbenoid levels. These findings have provided a new approach to producing glycosidic compounds and corresponding aglycones in cell culture.

     

Involvement of kinetin and spermidine in controlling salinity stress in mulberry (Morus alba L. cv. S1)

In mulberry (Morus alba L.) plants NaCl stress imposed through roots by irrigation during growth period decreased the net photosynthetic rate (NPR), physiological water use efficiency (pWUE), which ultimately reflected on the reduction of growth parameters and leaf yield. Foliar spray of kinetin and spermidine (both at 1 mM) on salinized plants reduced the detrimental effects of saline stress. Kinetin and spermidine sprayed plants increased the total chlorophyll, protein content, as well as leaf yield, but reduced the sugar and proline contents as compared to NaCl treated plants. Kinetin was more effective than spermidine in increasing NPR, pWUE and leaf yield both in nonsalinized and salinized condition.     

Apical dominance in mulberry (Morus alba): Effects of position of lateral and accessory buds and leaves

Suzuki, T. 1990. Apical dominance in mulberry ( Morus alba ): Effects of position of lateral and accessory buds and leaves. – Physiol. Plant. 78: 468-474.
Removing apical portions of current growth coppice shoots from field-grown, low-pruned stumps of mulberry ( Morus alba L. cv. Shin-ichinose) caused sprouting of one or more upper main buds, almost concurrently with that of accessory buds. However, removal of the new sprouts, including those from accessory buds, slightly enhanced the sprouting of buds immediately below them, and did not affect buds lower down. In contrast, mature leaves inhibited the buds in their axils. Budless, leafy nodes on the upper part of pruned shoots tended to swell after treatment, perhaps due to the accumulation of substances translocated from the roots and possibly from the remaining leaves. Lateral buds at different positions along the shoot differed in their sprouting ability with buds lower on the shoot being more inhibited. This inhibition gradient dissappeared when all coppice shoots on one stump were pruned to the same bud position, suggesting inhibition from neighboring, actively growing shoots. These results demonstrate that acropetal influences are important in bud dominance relationships.     

Ameliorative effects of mulberry (Morus alba L.) leaves on hyperlipidemia in rats fed a high-fat diet: induction of fatty acid oxidation, inhibition of lipogenesis, and suppression of oxidative stress

To determine the effects of mulberry (Morus alba L.) leaves on hyperlipidemia, we performed gene expression profiling of the liver. Rats were fed a high-fat diet and administered mulberry leaves for 7 weeks. Plasma triglyceride and non-esterified fatty acid levels were significantly lower in the rats treated with mulberry leaves as compared with the untreated rats. DNA microarray analysis revealed that mulberry leaves upregulated expression of the genes involved in α-, β- and ω-oxidation of fatty acids, mainly related to the peroxisome proliferator-activated receptor signaling pathway, and downregulated the genes involved in lipogenesis. Furthermore, treatment with mulberry leaves upregulated expression of the genes involved in the response to oxidative stress. These results indicate that consumption of fatty acids and inhibition of lipogenesis are responsible for the reduction in plasma lipids caused by mulberry administration. In addition, mulberry treatment maintains the body's oxidative state at a low level despite enhancing fatty acid oxidation.