Long term exogenous putrescine application improves grain yield of a salt-sensitive rice cultivar exposed to NaCl

In order to determine the impact of polyamines on the yield-related parameters of rice (Oryza sativa L.) exposed to NaCl, the plants belonging to a salt-sensitive cultivar I Kong Pao were maintained from the young seedling stage until harvest on nutrient solutions containing 0 or 30 mM NaCl in the presence or absence of 10 μM putrescine (Put), 10 μM spermidine (Spd) or 10 μM spermine (Spm). Exogenous Put and to a lesser extent exogenous Spd improved growth and yield of salt-treated plants in relation to an increase in K⁺/Na⁺ ratio of shoots and roots as compared to plants exposed to NaCl in the absence of exogenous polyamines. Exogenous Put also improved the net CO₂ assimilation, at least partly as a consequence of an increase in the stomatal conductance. Yield increase of salt-treated plants exposed to Put was related to an improvement of floral morphogenesis leading to a higher number of fertile tillers per plant and a higher number of spikelets per panicle. Putrescine also improved the pollen viability in salt-treated plants, allowing a higher seed set and thus a higher grain yield per plant. Although polyamines accumulated in the shoots to some extent in response to exogenous application, neither Put nor Spd accumulated in the seeds. In contrast, Spm did not afford any protection of salt-treated plants but was translocated to the seeds during maturation. Seeds with a high internal Spm concentration exhibited delayed germination in the presence of NaCl. These data are discussed in relation to the implication of polyamine in the metabolism and physiology of salt-treated plants.     

Exogenously applied polyamines increase drought tolerance of rice by improving leaf water status,photosynthesis and membrane properties

Drought stress hampers rice performance principally by disrupting the plant–water relations and structure of biological membranes. This study appraised the role of polyamines (PAs) in improving drought tolerance in fine grain aromatic rice (Oryza sativa L.). Three PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)] were used each at 10 μM as seed priming (by soaking seeds in solution) and foliar spray. Primed and non-primed seeds were sown in plastic pots with normal irrigation in a phytotron. At four-leaf stage, plants were subjected to drought stress by bringing the soil moisture down to 50% of field capacity by halting water supply. For foliar application, 10 μM solutions each of Put, Spd and Spm were sprayed at five-leaf stage. Results revealed that drought stress severely reduced the rice fresh and dry weights, while PAs application improved net photosynthesis, water use efficiency, leaf water status, production of free proline, anthocyanins and soluble phenolics and improved membrane properties. PAs improved drought tolerance in terms of dry matter yield and net photosynthesis was associated with the maintenance of leaf water status and improved water use efficiency. Among the antioxidants, catalase activity was negatively related to H₂O₂ and membrane permeability, which indicated alleviation of oxidative damage on cellular membranes by PAs application. Foliar application was more effective than the seed priming, and among the PAs, Spm was the most effective in improving drought tolerance.     

Involvement of polyamines in the drought resistance of rice

This study investigated whether and how polyamines (PAs) in rice (Oryza sativa L.) plants are involved in drought resistance. Six rice cultivars differing in drought resistance were used and subjected to well-watered and water-stressed treatments during their reproductive period. The activities of arginine decarboxylase, S-adenosyl-L-methionine decarboxylase, and spermidine (Spd) synthase in the leaves were significantly enhanced by water stress, in good agreement with the increase in putrescine (Put), Spd, and spermine (Spm) contents there. The increased contents of free Spd, free Spm, and insoluble-conjugated Put under water stress were significantly correlated with the yield maintenance ratio (the ratio of grain yield under water-stressed conditions to grain yield under well-watered conditions) of the cultivars. Free Put at an early stage of water stress positively, whereas at a later stage negatively, correlated with the yield maintenance ratio. No significant differences were observed in soluble-conjugated PAs and insoluble-conjugated Spd and Spm among the cultivars. Free PAs showed significant accumulation when leaf water potentials reached -0.51 MPa to -0.62 MPa for the drought-resistant cultivars and -0.70 MPa to -0.84 MPa for the drought-susceptible ones. The results suggest that rice has a large capacity to enhance PA biosynthesis in leaves in response to water stress. The role of PAs in plant defence to water stress varies with PA forms and stress stages. In adapting to drought it would be good for rice to have the physiological traits of higher levels of free Spd/free Spm and insoluble-conjugated Put, as well as early accumulation of free PAs, under water stress.     

Waterlogging tolerance of Welsh onion (Allium fistulosum L.) enhanced by exogenous spermidine and spermine

Soil flooding is a seasonal factor that negatively affects plant performance and crop yields. In order to investigate the effects of spermidine (Spd) and spermine (Spm) on the waterlogging stress, it was checked that the content of relative water content (RWC), proline, chlorophyll and malondialdehyde (MDA), net photosynthesis, the rate of hydrogen peroxide (H₂O₂) and superoxide radicals (O₂^?) generation and the antioxidant enzyme activities of superoxide dismutase (SOD) (EC 1.15.1.1), catalase (CAT) (EC 1.11.1.6), ascorbate peroxidase (APX) (EC 1.11.1.11) and glutathione reductase (GR) (EC 1.6.4.2) in Welsh onion (Allium fistulosum L) plants. Pretreatment with 2 mM of Spd and Spm effectively maintained the balance of water content in plant leaves and roots under flooding stress. In addition, the data indicate that the protective role of proline should be considered minimal, as its accumulation was found to be inversely correlated with tolerance to stress; it also significantly retarded the loss of chlorophyll, enhanced photosynthesis, decreased the rate of O₂^? generation and H₂O₂ content, and prevented flooding-induced lipid peroxidation. Spd and Spm helped to maintain antioxidant enzyme activities under flooding; however, APX activity was found to increase slightly in response to Spm. The antioxidant system, an important component of the water-stress-protective mechanism, can be changed by PAs, which are able to moderate the radical scavenging system and to lessen in this way the oxidative stress. The results suggest that pretreatment with Spd and Spm prevents oxidative damage, and the protective effect of Spd was found to be greater than that of Spm.     

Polyamine and ethylene interactions in grain filling of superior and inferior spikelets of rice

This study was to test the hypothesis that polyamines (PAs) and ethylene and their interactions may be involved in mediating the post-anthesis development of spikelets in rice (Oryza sativa L.). Six rice cultivars differing in grain filling rate were field-grown, and the changing patterns of PAs and ethylene levels in rice spikelets during the filling and their relations with grain filling rates were investigated. The results showed that inferior spikelets had much greater ethylene evolution rate and 1-aminocylopropane-1-carboxylic acid (ACC) concentration than superior spikelets. Opposite to ethylene production, superior spikelets showed much higher free-spermidine (Spd) and free-spermine (Spm) concentrations than inferior spikelets. Grain filling rate was very significantly and negatively correlated with ethylene evolution rate and ACC concentration, whereas positively correlated with free-Spd and free-Spm concentrations and with the ratio of free-Spd or free-Spm to ACC. Application of Spd, Spm, or aminoethoxyvinylglycine (an inhibitor of ethylene synthesis by inhibiting ACC synthesis) to panicles at the early grain filling stage significantly reduced ethylene evolution rate and ACC concentration, while significantly increased Spd and Spm concentrations, grain filling rate and grain weight of inferior spikelets. Application of ACC, ethephon (an ethylene-releasing agent), or methylglyoxal-bis (guanylhydrazone) (an inhibitor of Spd and Spm synthesis) showed the opposite effects. The results suggest that antagonistic interactions between PAs (Spd and Spm) and ethylene may be involved in mediating grain filling. A higher ratio of free-Spd or free-Spm to ethylene in rice spikelets could enhance grain filling.     

Polyamines and ethylene in rice young panicles in response to soil drought during panicle differentiation

This study tested the hypothesis that polyamines (PA) and ethylene (ETH) mediate the effects of soil drought on spikelet development in rice (Oryza sativa L.). Two rice cultivars, Yong You-2640 and Yang Dao-6, with vastly different panicle sizes were grown in pots under three soil moisture treatments: well-watered (WW), moderate soil drought (MD) and severe soil drought (SD), from the onset of panicle initiation to the pollen completion stage. MD treatment significantly increased spikelet differentiation, spikelet number per panicle, fully filled grain percentage and grain yield, decreasing the percentage of degenerated spikelets, sterile spikelets and partially filled grains compared to WW treatment. In contrast, SD treatment showed opposite effects. MD also increased the contents of free spermidine (Spd), free spermine (Spm) and the ratios of free putrescine, free-Spd and free-Spm to 1-aminocylopropane-1-carboxylic acid (ACC), decreasing the ETH evolution rate and ACC content in young panicles. In contrast, SD treatment showed opposite effects. Furthermore, free-Spd and free-Spm contents increased significantly, while ETH and ACC levels, and the percentage of degenerated and sterile spikelets decreased significantly under application of Spd or an inhibitor of ETH synthesis. The results were reversed when ACC or an inhibitor of Spd and Spm synthesis was applied. These findings suggest antagonistic interactions between free-PA (Spd and Spm) and ETH in response to soil drought, mediating spikelet development in rice.     

Growth characteristics and endosperm structure of superior and inferior spikelets of indica rice under high-temperature stress

Heat stress severely reduces rice yield and quality; however, differences between the superior, early-flowering and inferior, later-flowering spikelets of indica rice in response to high-temperature stress during grain filling remain unclear. This study investigated the effects of high temperature (HT, 33.6/20.7 °C day/night) on growth, endosperm structure, and hormone and polyamine content of superior and inferior spikelets of heat-sensitive (SG-1) and heat-tolerant (HHZ) indica cultivars. The HT decreased fertilization rate, caused earlier grain filling, and reduced duration of grain filling, thus resulting in decreased grain mass and a poor endosperm structure. In addition, soluble sugar and sucrose content increased, and starch synthesis decreased by HT at the early stage of grain filling. The HT increased polyamine [spermidine (Spd) and spermine (Spm)] and abscisic acid (ABA) content, but reduced zeatin (Z) + zeatin riboside (ZR) and indole-3-acetic acid (IAA) content in the grains. Such effects were more apparent in the inferior than superior spikelets; however, the inferior spikelets of SG-1 were more affected than those of HHZ. At the middle grain filling stage, HT produced little difference between the two cultivars. Our results suggest that the poor development of inferior spikelets of SG-1 under the HT could be attributed, at least in part, to the changed content and ratios of free polyamines [putrescine (Put), Spd, and Spm] and phytohormones (Z+ZR, IAA, and ABA) and the conversion efficiency of sucrose into starch.     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

Seed-specific silencing of OsMRP5 reduces seed phytic acid and weight in rice

Phytic acid (PA) is poorly digested by humans and monogastric animals and negatively affects human/animal nutrition and the environment. Rice mutants with reduced PA content have been developed but are often associated with reduced seed weight and viability, lacking breeding value. In the present study, a new approach was explored to reduce seed PA while attaining competitive yield. The OsMRP5 gene, of which mutations are known to reduce seed PA as well as seed yield and viability, was down-regulated specifically in rice seeds by using an artificial microRNA driven by the rice seed specific promoter Ole18. Seed PA contents were reduced by 35.8–71.9 % in brown rice grains of transgenic plants compared to their respective null plants (non-transgenic plants derived from the same event). No consistent significant differences of plant height or number of tillers per plant were observed, but significantly lower seed weights (up to 17.8 % reduction) were detected in all transgenic lines compared to null plants, accompanied by reductions of seed germination and seedling emergence. It was observed that the silencing of the OsMRP5 gene increased the inorganic P (Pi) levels (up to 7.5 times) in amounts more than the reduction of PA-P in brown rice. This indicates a reduction in P content in other cellular compounds, such as lipids and nucleic acids, which may affect overall seed development. Put together, the present study demonstrated that seed specific silencing of OsMRP5 could significantly reduce the PA content and increase Pi levels in seeds; however, it also significantly lowers seed weight in rice. Discussions were made regarding future directions towards producing agronomically competitive and nutritionally valuable low PA rice.     

虫压胁迫对抗虫转基因水稻生长发育及产量性状的影响

在大田条件下,以转基因抗虫水稻Bt63、R1和R2及非转基因水稻汕优63(对照)为材料,设置高、低两种虫压环境条件,研究虫压胁迫对转Bt抗虫基因水稻生长发育及产量相关性状的影响.结果表明： 抗虫水稻在虫压胁迫条件下可充分体现出外源基因的抗性特点.在高虫压条件下,3种转Bt基因水稻受螟虫危害程度远低于对照植株,株高、分蘖数、地上部鲜质量、穗长、穗质量、单株穗数、单株实粒数、实粒质量、结实率、千粒重等生长发育和产量指标均高于对照,但仅株高、分蘖数和穗长3个指标与对照有显著差异.因此,抗虫外源Bt基因的引入对水稻结实性不会产生负面效应,高虫压胁迫条件对抗虫转基因水稻产量的影响较小.     

Brassinosteroids promote development of rice pollen grains and seeds by triggering expression of Carbon Starved Anther,a MYB domain protein

Transport of photoassimilates from leaf tissues (source regions) to the sink organs is essential for plant development. Here, we show that a phytohormone, the brassinosteroids (BRs) promotes pollen and seed development in rice by directly promoting expression of Carbon Starved Anther (CSA) which encodes a MYB domain protein. Over‐expression of the BR‐synthesis gene D11 or a BR‐signaling factor OsBZR1 results in higher sugar accumulation in developing anthers and seeds, as well as higher grain yield compared with control non‐transgenic plants. Conversely, knockdown of D11 or OsBZR1 expression causes defective pollen maturation and reduced seed size and weight, with less accumulation of starch in comparison with the control. Mechanically, OsBZR1 directly promotes CSA expression and CSA directly triggers expression of sugar partitioning and metabolic genes during pollen and seed development. These findings provide insight into how BRs enhance plant reproduction and grain yield in an important agricultural crop.     

Brassinosteroids regulate grain filling in rice

Genes controlling hormone levels have been used to increase grain yields in wheat (Triticum aestivum) and rice (Oryza sativa). We created transgenic rice plants expressing maize (Zea mays), rice, or Arabidopsis thaliana genes encoding sterol C-22 hydroxylases that control brassinosteroid (BR) hormone levels using a promoter that is active in only the stems, leaves, and roots. The transgenic plants produced more tillers and more seed than wild-type plants. The seed were heavier as well, especially the seed at the bases of the spikes that fill the least. These phenotypic changes brought about 15 to 44% increases in grain yield per plant relative to wild-type plants in greenhouse and field trials. Expression of the Arabidopsis C-22 hydroxylase in the embryos or endosperms themselves had no apparent effect on seed weight. These results suggested that BRs stimulate the flow of assimilate from the source to the sink. Microarray and photosynthesis analysis of transgenic plants revealed evidence of enhanced CO₂ assimilation, enlarged glucose pools in the flag leaves, and increased assimilation of glucose to starch in the seed. These results further suggested that BRs stimulate the flow of assimilate. Plants have not been bred directly for seed filling traits, suggesting that genes that control seed filling could be used to further increase grain yield in crop plants.     

Putrescine overproduction does not affect the catabolism of spermidine and spermine in poplar and Arabidopsis

The effect of up-regulation of putrescine (Put) production by genetic manipulation on the turnover of spermidine (Spd) and spermine (Spm) was investigated in transgenic cells of poplar (Populus nigra × maximowiczii) and seedlings of Arabidopsis thaliana. Several-fold increase in Put production was achieved by expressing a mouse ornithine decarboxylase cDNA either under the control of a constitutive (in poplar) or an inducible (in Arabidopsis) promoter. The transgenic poplar cells produced and accumulated 8–10 times higher amounts of Put than the non-transgenic cells, whereas the Arabidopsis seedlings accumulated up to 40-fold higher amounts of Put; however, in neither case the cellular Spd or Spm increased consistently. The rate of Spd and Spm catabolism and the half-life of cellular Spd and Spm were measured by pulse-chase experiments using [¹⁴C]Spd or [¹⁴C]Spm. Spermidine half-life was calculated to be about 22–32 h in poplar and 52–56 h in Arabidopsis. The half-life of cellular Spm was calculated to be approximately 24 h in Arabidopsis and 36–48 h in poplar. Both species were able to convert Spd to Spm and Put, and Spm to Spd and Put. The rates of Spd and Spm catabolism in both species were several-fold slower than those of Put, and the overproduction of Put had only a small effect on the overall rates of turnover of Spd or Spm. There was little effect on the rates of Spd to Spm conversion as well as the conversion of Spm into lower polyamines. While Spm was mainly converted back to Spd and not terminally degraded, Spd was removed from the cells largely through terminal catabolism in both species.     

Ectopic expression of PgRab7 in rice plants (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) results in differential tolerance at the vegetative and seed setting stage during salinity and drought stress

In this work, we have overexpressed a vesicle trafficking protein, Rab7, from a stress-tolerant plant, Pennisetum glaucum, in a high-yielding but stress-sensitive rice variety Pusa Basmati-1 (PB-1). The transgenic rice plants were tested for tolerance against salinity and drought stress. The transgenic plants showed considerable tolerance at the vegetative stage against both salinity (200 mM NaCl) and drought stress (up to 12 days after withdrawing water). The protection against salt and drought stress may be by regulating Na⁺ ion homeostasis, as the transgenic plants showed altered expression of multiple transporter genes, including OsNHX1, OsNHX2, OsSOS1, OsVHA, and OsGLRs. In addition, decreased generation and maintenance of lesser reactive oxygen species (ROS), with maintenance of chloroplast grana and photosynthetic machinery was observed. When evaluated for reproductive growth, 89–96 % of seed setting was maintained in transgenic plants during drought stress; however, under salt stress, a 33–53 % decrease in seed setting was observed. These results indicate that PgRab7 overexpression in rice confers differential tolerance at the seed setting stage during salinity and drought stress and could be a favored target for raising drought-tolerant crops.     

Spermidine alleviates the growth of saline-stressed ginseng seedlings through antioxidative defense system

Protective effects of exogenous spermidine (Spd), activity of antioxygenic enzymes, and levels of free radicals in a well-known medicinal plant, Panax ginseng was examined. Seedlings grown in salinized nutrient solution (150 mM NaCl) for 7 d exhibited reduced relative water content, plant growth, increased free radicals, and showing elevated lipid peroxidation. Application of Spd (0.01, 0.1, and 1 mM) to the salinized nutrient solution showed increased plant growth by preventing chlorophyll degradation and increasing PA levels, as well as antioxidant enzymes such as CAT, APX, and GPX activity in the seedlings of ginseng. During salinity stress, Spd was effective for lowering the accumulation of putrescine (Put), with a significant increase in the spermidine (Spd) and spermine (Spm) levels in the ginseng seedlings. A decline in the Put level ran parallel to the higher accumulation of proline (Pro), and exogenous Spd also resulted in the alleviation of Pro content under salinity. Hydrogen peroxide (H₂O₂) and superoxide (O₂⁻) production rates were also reduced in stressed plants after Spd treatment. Furthermore, the combined effect of Spd and salt led to a significant increase in diamine oxidase (DAO), and subsequent decline in polyamine oxidase (PAO). These positive effects were observed in 0.1 and 1 mM Spd concentrations, but a lower concentration (0.01 mM) had a very limited effect. In summary, application of exogenous Spd could enhance salt tolerance of P. ginseng by enhancing the activities of enzyme scavenging system, which influence the intensity of oxidative stress.     

Homologous expression of γ-glutamylcysteine synthetase increases grain yield and tolerance of transgenic rice plants to environmental stresses

Various environmental stresses induce reactive oxygen species (ROS), causing deleterious effects on plant cells. Glutathione (GSH), a critical antioxidant, is used to combat ROS. GSH is produced by γ-glutamylcysteine synthetase (γ-ECS) and glutathione synthetase (GS). To evaluate the functional roles of the Oryza sativa L. Japonica cv. Ilmi ECS (OsECS) gene, we generated transgenic rice plants overexpressing OsECS under the control of an inducible promoter (Rab21). When grown under saline conditions (100 mM) for 4 weeks, 2-independent transgenic (TGR1 and TGR2) rice plants remained bright green in comparison to control wild-type (WT) rice plants. TGR1 and TGR2 rice plants also showed a higher GSH/GSSG ratio than did WT rice plants in the presence of 100 mM NaCl, which led to enhanced redox homeostasis. TGR1 and TGR2 rice plants also showed lower ion leakage and higher chlorophyll-fluorescence when exposed to 10 μM methyl viologen (MV). Furthermore, the TGR1 and TGR2 rice seeds had approximately 1.5-fold higher germination rates in the presence of 200 mM salt. Under paddy field conditions, OsECS-overexpression in transgenic rice plants increased rice grain yield (TGW) and improved biomass. Overall, our results show that OsECS overexpression in transgenic rice increases tolerance and germination rate in the presence of abiotic stress by improving redox homeostasis via an enhanced GSH pool. Our findings suggest that increases in grain yield by OsECS overexpression could improve crop yields under natural environmental conditions.