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Singlet oxygen (1O2) has a very short half-life of 10−5 s; however, it is a strong oxidant that causes growth arrest and necrotic lesions on plants1–4. Its signaling pathway remains largely unknown. The Arabidopsis flu (FLUORESCENT) mutant accumulates a high level of 1O2 and shows drastic changes in nuclear gene expression5. Only two plastid proteins, EX1 (EXECUTER 1) and EX2 (EXECUTER 2), have been identified in the singlet oxygen signaling6,7. Here we found that the transcription factor ABI4 (ABSCISIC ACID INSENSITIVE 4) binds the promoters of genes responsive to 1O2-signals. Inactivation of the ABI4 protein in the flu/abi4 double mutant was sufficient to suppress the upregulation of almost all 1O2-responsive-genes and rescued the lethal phenotype of flu grown under light / dark cycles, similar to the flu/ex1/ex2 triple mutant. In addition to cell death, we reported for the first time that 1O2 also induces cell wall thickening and stomatal development defect. However, no apparent growth arrest was observed for the flu mutant under normal light / dim light cycles, but the cell wall thickening (doubled) and stomatal reduction (by two-thirds) still occurred. These results offer a new idea for breeding stress tolerant and lodging resistant varieties in agricultural industry.

Intensively farmed crops used to experience numerous environmental stresses. Among these, shade and drought significantly influence the morpho-physiological and biochemical attributes of plants. However, the interactive effect of shade and drought on the growth and development of soybean under dense cropping systems has not been reported yet. This study investigated the interactive effect of PEG-induced osmotic stress and shade on soybean seedlings. The soybean cultivar viz., C-103 was subjected to PEG-induced osmotic stress from polyethylene glycol 6000 (PEG-6000) under shading and non-shading conditions. PEG-induced osmotic stress significantly reduced the relative water contents, morphological parameters, carbohydrates and chlorophyll contents under both light environments. A significant increase was observed in osmoprotectants, reactive oxygen species and antioxidant enzymes in soybean seedlings. Henceforth, the findings revealed that, seedlings grown under non-shading conditions produced more malondialdehyde and hydrogen peroxide contents as compared to the shade-treated plants when subjected to PEG-induced osmotic stress. Likewise, the shaded plants accumulated more sugars and proline than non-shaded ones under drought stress. Moreover, it was found that non-shaded grown plants were more sensitive to PEG-induced osmotic stress than those exposed to shading conditions, which suggested that shade could boost the protective mechanisms against osmotic stress or at least would not exaggerate the adverse effects of PEG-induced osmotic stress in soybean seedlings.

When a plant is exposed to heterogeneous light, the photosynthesis of unshaded leaves is often stimulated to compensate for the decline in photosynthesis of shaded leaves, i.e., photosynthetic compensation. However, a decline of photosynthesis in unshaded leaves, which means an impairment of photosynthetic compensation, has also been widely reported. Herein, two cultivars of maize (Zea mays L.), 'Rongyu1210' (RY) and 'Zhongdan808' (ZD), were studied comparatively. Both cultivars performed evident photosynthetic compensation under heterogeneous light (HL) as the light phase begins (8:30 a.m.). However, as the light phase continues (10:30 a.m.), an impairment of photosynthetic compensation took place in HL-treated ZD, but not in HL-treated RY. For both cultivars, nitrogen content of unshaded leaves was higher under HL, indicating a preferential nitrogen distribution towards unshaded leaves. This is related to the photosynthetic compensation but not the cause of the impairment. In addition, no obvious difference was found in the response of photosynthates (sucrose and starch) to HL between cultivars at 8:30 a.m. However, at 10:30 a.m., the content of photosynthates decreased significantly in unshaded leaves of HL-treated RY, along with increased abundances of both sucrose transporters (SUTs) and H

Intercropping is one of the most vital practice to improve land utilization rate in China that has limited arable land resource. However, the traditional intercropping systems have many disadvantages including illogical field lay-out of crops, low economic value, and labor deficiency, which cannot balance the crop production and agricultural sustainability. In view of this, we developed a novel soybean strip intercropping model using maize as the partner, the regular maize-soybean strip intercropping mainly popularized in northern China and maize-soybean relay-strip intercropping principally extended in southwestern China. Compared to the traditional maize-soybean intercropping systems, the main innovation of field lay-out style in our present intercropping systems is that the distance of two adjacent maize rows are shrunk as a narrow strip, and a strip called wide strip between two adjacent narrow strips is expanded reserving for the growth of two or three rows of soybean plants. The distance between outer rows of maize and soybean strips are expanded enough for light use efficiency improvement and tractors working in the soybean strips. Importantly, optimal cultivar screening and increase of plant density achieved a high yield of both the two crops in the intercropping systems and increased land equivalent ratio as high as 2.2. Annually alternative rotation of the adjacent maize- and soybean-strips increased the grain yield of next seasonal maize, improved the absorption of nitrogen, phosphorus, and potasium of maize, while prevented the continuous cropping obstacles. Extra soybean production was obtained without affecting maize yield in our strip intercropping systems, which balanced the high crop production and agricultural sustainability.

Soybean [Glycine max (L.) Merr.] is a widely cultivated crop, whose growth and development are usually influenced by fluctuating light environment in fields. In the present study, two soybean cultivars (Nandou12 and Nan 032-4) were selected to study the regulatory mechanisms of capability to do photosynthetic work under different light intensities. Results showed that the net photosynthetic rate (P n ) of Nandou 12, which has a higher yield in field condition, was significantly higher than that of Nan 032-4 under all the light intensities studied. Measurements on stomatal conductance (G s) and intercellular CO2 concentration (C i) suggested that the difference of P n between these two cultivars was mainly caused by non-stomatal factors. Transcript detection found that the expression of genes encoding plastid terminal oxidase (PTOX) and alternative oxidase (AOX), which represent the terminal oxidases of chlororespiration in chloroplasts and alternative respiration in mitochondria, respectively, was up-regulated in both cultivars when light intensity increased, implying potential roles of these two terminal oxidases in photosynthetic regulation. Further analysis showed that the expression level of PTOX increased more obviously in Nandou 12, together with higher non-photochemical quenching (NPQ) and higher activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) than that in Nan 032-4, when light intensity increased. However, Nan 032-4 performed a more obvious up-regulation of AOX genes, as well as higher activities of NAD-malate dehydrogenase (MDH) and NADP-MDH than Nandou 12, when light intensity increased. These distinctions were consequently suggested to be potential reasons for the difference of photosynthetic performance between two soybean cultivars.

Receptor-like kinases BAK1 and BKK1 modulate multiple cellular processes including brassinosteroid signaling and PRR-mediated PTI in Arabidopsis. Our previous reports also demonstrated that bak1 bkk1 double mutants exhibit a spontaneous cell death phenotype under normal growth condition. With an unknown mechanism, the cell death in bak1 bkk1 is significantly suppressed when grown in dark but can be quickly induced by light. Furthermore, little is known about intrinsic components involved in BAK1 and BKK1-regulated cell death pathway. In this study, we analyzed how light functions as an initiator of cell death and identified ETI components to act as mediators of cell death signaling in bak1 bkk1. Cell death suppressed in bak1 bkk1 by growing in dark condition recurred upon exogenously treated SA. SA biosynthesis-related genes SID2 and EDS5, which encode chloroplast-localized proteins, were highly expressed in bak1-4 bkk1-1. When crossed to bak1-3 bkk1-1, sid2 or eds5 was capable of efficiently suppressing the cell death. It suggested that overly produced SA is crucial for inducing cell death in bak1 bkk1 grown in light. Notably, bak1-3 or bkk1-1 single mutant was shown to be more susceptible but bak1-3 bkk1-1 double mutant exhibited enhanced resistance to bacterial pathogen, suggesting immune signaling other than PTI is activated in bak1 bkk1. Moreover, genetic analyses showed that mutation in EDS1 or PAD4, key ETI mediator, significantly suppressed the cell death in bak1-3 bkk1-1. In this study, we revealed that light-triggered SA accumulation plays major role in inducing the cell death in bak1 bkk1, mediated by ETI components.

Nitric oxide (NO) is extensively involved in various growth processes and stress responses in plants; however, the regulatory mechanism of NO-modulated cellular sugar metabolism is still largely unknown. Here, we report that NO significantly inhibited monosaccharide catabolism by modulating sugar metabolic enzymes through S-nitrosylation (mainly by oxidizing dihydrolipoamide, a cofactor of pyruvate dehydrogenase). These S-nitrosylation modifications led to a decrease in cellular glycolysis enzymes and ATP synthase activities as well as declines in the content of acetyl coenzyme A, ATP, ADP-glucose and UDP-glucose, which eventually caused polysaccharide-biosynthesis inhibition and monosaccharide accumulation. Plant developmental defects that were caused by high levels of NO included delayed flowering time, retarded root growth and reduced starch granule formation. These phenotypic defects could be mediated by sucrose supplementation, suggesting an essential role of NO-sugar cross-talks in plant growth and development. Our findings suggest that molecular manipulations could be used to improve fruit and vegetable sweetness.

Physical characteristics of stem are closely relative to the crop lodging. Increase of stem strength is conducive to resolve the problem of lodging. Three soybean cultivars with different shade tolerance were planted under maize-soybean intercropping and soybean monocropping, respectively. Physiological and biochemical indices including cellulose, soluble sugar, sucrose, starch contents and enzyme activity were investigated to assess the snapping resistance and lodging resistance of the stems of soybean seedling, and snapping- and lodging-resistance indices were calculated for further verification. Furthermore, relationship analyses between these factors and the lodging of inter-cropped soybean showed that the intercropping soybean lodged seriously, the snapping resistance, lodging resistance index, contents of cellulose, soluble sugar, sucrose, starch and activities of the related enzymes were significantly lower than monocropping soybean at seedling stage. The three soybean cultivars showed different phenotypes in intercropping condition. The snapping-resistant Nandou12 with strong shade-tolerant traits was the most lodging-resistant phenotype, and it also harbored high contents of cellulose, soluble sugar, sucrose, starch and active enzymes. The lodging resistance index, cellulose content of the stems of intercropped soybean seedling were significantly positively correlated with the snapping resistance, and were significantly negatively correlated with the actual lodging percentage. The activities of sucrose phosphate synthase (SPS) , sucrose synthase (SS) and neutral invertase (NI) were positively correlated with sucrose is content, but not the acid invertase (AI). The activities of SPS, NI and SS were positively correlated with cellulose content, but not Al. In a word, the high activities of SPS and SS in the soybean stem were the enzymatic basis to maintain relatively higher cellulose and sucrose content, which is conducive to improve the stem-sfrength and enhance the lodging resistance ability in intercropping condition. Effects of different light conditions on cellulose metabolic mechanism of soybean seedling stems, lodging resistant characteristics of soybean seedlings studied in the corn-soybean intercropping system provided a basis for screening more shade-tolerant soybean variety.

Cadmium (Cd) has been identified as a significant pollutant due to its high solubility in water and soil and high toxicity to plants and animals. Rice, as one of the most important food crops, is grown in soils with variable levels of Cd and therefore, is important to discriminate the Cd tolerance of different rice cultivars to determine their suitability for cultivation in Cd-contaminated soils. This study investigates the primary mechanisms employed by four rice cultivars in attaining Cd tolerance. HA63 cultivar reduces Cd uptake by increasing Fe absorption through activation of phytosiderophores. T3028 cultivar accumulates the highest level of Cd in leaves while also activating its reactive oxygen species (ROS) scavenging system, including antioxidant enzymes and phytochelatins. In some rice cultivars (such as HA63), a cyanide-resistant respiration mechanism, important in Cd detoxification, was also promoted under the Cd stress. In conclusion, different rice cultivars may adopt different biochemical strategies and respond with different efficiency to Cd stress.

The effects of n-propyl gallate (nPG) on postharvest ripening of tomato have been studied. Mature green (MG) stage tomatoes were harvest and treated with 0.5 mM nPG at 25 ± 1C for 12 h. Following the nPG treatment, fruit ripening was delayed by at least 7 days. Cyanide-resistant respiration and ethylene production were suppressed significantly at the first 7 days. nPG treatment decreased levels of H2O2 and increased activities of antioxidant enzymes and chemicals, particularly for superoxide dismutase (SOD), catalase (CAT) and guaiacol peroxidase (GPX) and ascorbic acid (AsA). The decreases of flesh firmness and total chlorophylls were inhibited, and the production of soluble sugars and lycopene were suppressed by nPG either. Alternative oxidase (AOX) and cyanide-resistant respiration play important roles in fruit climacteric and may be correlated with system 2 ethylene production. nPG could be used as an efficient inhibitor to tomato fruit ripening. PRACTICAL APPLICATIONS Control of fruit ripening is of enormous commercial importance. Generally, tomato fruit ripening is controlled through the use of gas, temperature and humidity control. This research has focused on the application of n-propyl gallate (nPG) on postharvest ripening of tomato fruit. The results of the study have implied that nPG treatment could obviously prolong the shelf life and keep the postharvest quality of tomato fruits, and the firmness loss, respiration rate and ethylene production could be better suppressed compared with the control fruits, which shows that nPG is a potentially effective and safe inhibitor for climacteric fruits ripening, such as tomato. Utilization of the results of this research is likely to benefit for the storage technological improvement.

Mitochondrial alternative oxidase (AOX), the unique respiratory terminal oxidase in plants, catalyzes the energy wasteful cyanide (CN)-resistant respiration and plays a role in optimizing photosynthesis. Although it has been demonstrated that leaf AOX is upregulated after illumination, the in vivo mechanism of AOX upregulation by light and its physiological significance are still unknown. In this report, red light and blue light-induced AOX (especially AOX1a) expressions were characterized. Phytochromes, phototropins and cryptochromes, all these photoreceptors mediate the light-response of AOX1a gene. When aox1a mutant seedlings were grown under a high-light (HL) condition, photobleaching was more evident in the mutant than the wild-type plants. More reactive oxygen species (ROS) accumulation and inefficient dissipation of chloroplast reducing-equivalents in aox1a mutant may account for its worse adaptation to HL stress. When etiolated seedlings were exposed to illumination for 4 h, chlorophyll accumulation was largely delayed in aox1a plants. We first suggest that more reduction of the photosynthetic electron transport chain and more accumulation of reducing-equivalents in the mutant during de-etiolation might be the main reasons.

Ligustrum vicaryi L. is a hybrid of Ligustrum ovalifolium Hassk. var. aureo-marginatum and Ligustrum vulgale L., and displays a chlorophyll-less phenotype. Therefore it is widely used as a horticultural shrub because of its golden-color leaves. Its putative mechanism, light responses, chlorophyll synthesis and plastid development were studied. L. vicaryi has a higher level of 5-aminolevulinic acid (ALA), but lower levels of chlorophylls compared with L. quihoui. The yellowish phenotype of L. vicaryi upper leaves could be attributed to their hampered conversion from chlorophyllide into chlorophyll a. Despite the enhanced ALA level and the decreased thylakoid stacking in plastids, L. vicaryi golden leaves contain normal levels of Lhcb transcripts and photosystem apoproteins. Furthermore, reactive oxygen species (ROS) accumulation is almost the same in L. vicaryi and L. quihoui. The golden leaves often turn green and the contents of chlorophylls increase with decreasing light intensity. Dynamic changes of chlorophyll-synthesis-system under the light transition were also analyzed.

Drought, salinity and cold are the major environmental factors impacting on survival and productivity of Tibetan hulless barley in Tibetan Plateau of China. Tibetan hulless barley cultivar, Tibetan Heiqingke No. 1, has developed a strong tolerance and adaptation to stresses in relation to the wild barley. The differences of dehydrin gene transcription and translation between Tibetan Heiqingke No. 1 and the wild barley under drought, salinity and low temperature stresses were investigated in the present study to figure out the putative mechanism of stress tolerance of Tibetan Heiqingke No. 1. The leaf relative water contents (RWCs) decreased more slowly in Tibetan hulless barley than the wild barley under osmotic and low temperature conditions. Electrolyte leakage, malondialdehyde and H2O2 contents increased faster in wild barley than those of Tibetan hulless barley, which indicated that cells of wild barley received more damages than Tibetan hulless barley. Furthermore, the expression of several dehydrin genes, belonging to four different classifications respectively, was also investigated. Polyclonal antibodies against dehydrins were obtained from rabbit after prokaryotic expression and purification of TDHN4, a dehydrin protein from Tibetan hulless barley. With these antibodies and dehydrin gene fragments, western blotting analysis and RT-PCR showed that Tibetan Heiqingke No. 1 accumulated higher abundance of dehydrins than stress-sensitive wild barley under all stress conditions.

BAK1 and BKK1 are receptor-like protein kinases (RLKs) involved in brassinosteroid signal transduction and plant resistance to bacteria and fungi. Here we report that loss-of-function mutants of BAK1 or BKK1 in Col-0 showed enhanced susceptibility to Turnip crinkle virus (TCV) infection. Cell death and chlorosis occurred much earlier in bak1-4 and bkk1-1 mutants than in wild-type plants, suggesting an important role of BAK1 and BKK1 in controlling cell death associated with TCV infection. The bak1-4 and bkk1-1 mutants showed the elevated transcription levels of pathogenesis genes, such as PR1, PR2, PR5, PDF1.2. Enhanced expression of these genes, however, failed to resist the transport of TCV virus. At 9 days postinoculation, TCV levels were the highest in the systemic leaves of bak1-4, but were undetected in BAK1 or BKK1 overexpressing plants. These results signify that both BAK1 and BKK1 are important components in controlling TCV infection in Arabidopsis plants.

The effects of exogenous salicylic acid (SA) on photosystem 2 (PS 2) in barley (Hordeum vulgare L.) seedlings were investigated. SA pretreatment provided protection against subsequent osmotic stress. The highest protective effect of 0.25 mM SA was confirmed by determination of chlorophyll fluorescence, electrolyte leakage, malonyldialdehyde contents, PS 2 mRNAs and proteins. SA pretreatment increased reactive oxygen species (ROS), decreased net photosynthetic rate and stomatal conductance immediately, but prevented ROS accumulation during subsequent osmotic stress by activating antioxidant enzymes. Elimination of H2O2 during SA pretreatment inhibited almost all above mentioned SA effects. Therefore, SA pretreatment enhanced osmotic stress tolerance in barley seedlings mainly through ROS signals, rather than SA itself. The only SA-dependent and ROS-independent effect of exogenous SA on PS 2 was reduction of non-photochemical quenching.

We searched and analyzed cyanobacteria-originated metazoa/fungi proteins (COPs) by phylogenetic analyses. Analysis of them showed that for millions of years universal plastid endosymbiosis and gene transfer occurred in ancestors of metazoa/fungi, and some transferred fragments have been reserved until now even in modern mammals. Most eukaryotes contained plastids in the ancient era, and some of them lost them later. Functions of homologues in cyanobacterial genomes and eukaryotic genomes are in consensus, and most are involved in the organic compound metabolism. With emergence of organelles and subcellular structure in eukaryotic cells, the locations of these proteins diversified. Furthermore, some novel functions were endowed for COPs, especially in vertebrates.

NYB is chlorophyll-less barley mutant, which is controlled by a recessive nuclear gene. The mutation mechanism is revealed. The activities of enzymes transforming 5-aminolevulinic acid into protochlorophyllide were the same in both NYB and the wild type (WT), but the activity of the protochlorophyllide oxidoreductase (POR) in WT was much higher than that of NYB. Most of the photosystem 2 apoproteins were present in both WT and NYB, suggesting that the capability of protein synthesis was probably fully preserved in the mutant. Thus chlorophyll (Chl) biosynthesis in NYB was hampered at conversion form protochlorophyllide (Pchlide) into chlorophyllide. The open reading frame of porB gene in NYB was inserted with a 95 bp fragment, which included a stop codon. The NYB mutant is a very useful material for studies of Chl biosynthesis, chloroplast signalling, and structure of light-harvesting POR-Pchlide complex (LHPP).

BRI1-ASSOCIATED KINASE 1 (BAK1) was initially identified as a co-receptor of the brassinosteroid (BR) receptor BRI1. Genetic analyses also revealed that BAK1 and its closest homolog BAK1-LIKE 1 (BKK1) regulate a BR-independent cell-death control pathway. The double null mutant bak1 bkk1 displays a salicylic acid- and light-dependent cell-death phenotype even without pathogen invasion. Molecular mechanisms of the spontaneous cell death mediated by BAK1 and BKK1 remain unknown. Here we report our identification of a suppressor of bak1 bkk1 (sbb1-1). Genetic analyses indicated that cell-death symptoms in a weak double mutant, bak1-3 bkk1-1, were completely suppressed by the loss-of-function mutation in SBB1, which encodes a nucleoporin (NUP) 85-like protein. Genetic analyses also demonstrated that individually knocking out three other nucleoporin genes from the SBB1-located sub-complex was also able to rescue the cell-death phenotype of bak1-3 bkk1-1. In addition, a DEAD-box RNA helicase, DRH1, was identified in the same protein complex as SBB1 via a proteomic approach. The drh1 mutation also rescues the cell-death symptoms of bak1-3 bkk1-1. Further analyses indicated that export of poly(A)(+) RNA was greatly blocked in the nup and drh1 mutants, resulting in accumulation of significant levels of mRNAs in the nuclei. Over-expression of a bacterial NahG gene to inactivate salicylic acid also rescues the cell-death phenotype of bak1-3 bkk1-1. Mutants suppressing cell-death symptoms always showed greatly reduced salicylic acid contents. These results suggest that nucleocytoplasmic trafficking, especially of molecules directly or indirectly involved in endogenous salicylic acid accumulation, is critical in BAK1- and BKK1-mediated cell-death control.

Field experiments were conducted in Shangluo pharmaceutical base in Shaanxi province to study the effect of nitrogen, phosphorus and potassium in different fertilization levels on Platycodon grandiflorum soil microorganism and activities of soil enzyme, using three-factor D-saturation optimal design with random block design. The results showed that N0P2K2, N2P2K0, N3P1K3 and N3P3K1 increased the amount of bacteria in 0-20 cm of soil compared with N0P0K0 by 144.34%, 39.25%, 37.17%, 53.58%, respectively. The amount of bacteria in 2040 cm of soil of N3P1K3 increased by 163.77%, N0P0K3 increased the amount of soil actinomycetes significantly by 192.11%, while other treatments had no significant effect. N2P0K2 and N3P1K3 increased the amounts of fungus significantly in 0-20 cm of soil compared with N0P0K0, increased by 35.27% and 92.21%, respectively. N3P0K0 increased the amounts of fungus significantly in 20-40 cm of soil by 165.35%, while other treatments had no significant effect. All treatments decrease soil catalase activity significantly in 0-20 cm of soil except for N2P0K2, and while N2P2K0 and NPK increased catalase activity significantly in 2040 cm of soil. Fertilization regime increased invertase activity significantly in 2040 cm of soil, and decreased phosphatase activity inordinately in 0-20 cm of soil, while increased phosphatase activity in 2040 cm of soil other than N1P3K3. N3P0K0, N0P0K3, N2P0K2, N2P2K0 and NPK increased soil urease activity significantly in 0-20 cm of soil compared with N0P0K0 by 18.22%, 14.87%,17.84%, 27.88%, 24.54%, respectively. Fertilization regime increased soil urease activity significantly in 2040 cm of soil other than N0P2K2.

Through the correlation analysis on the quantitative traits and their ratios of Salvia miltiorrhiza seedlings and seedling quality, a series of representative indices reflecting the seedling quality of the plant species were determined, and the seedling index suitable to the S. miltiorrhiza seedlings was ascertained by correlation degree analysis. Meanwhile, based on the relationships between the seedling index and the air temperature, solar radiation and air humidity, a simulation model for the seedling index of S. miltiorrhiza was established. The experimental data of different test plots and planting dates were used to validate the model. The results showed that the root diameter, stem diameter, crown dry mass, root dry mass, and plant dry mass had significant positive relationships with the other traits, and could be used as the indicators of the seedling's health. The seedling index of S. miltiorrhiza could be calculated by (stem diameter/root diameter + root dry mass/crown dry mass) x plant dry mass. The stem diameter, root dry mass, crown dry mass and plant dry mass had higher correlations with the seedling index, and thus, the seedling index determined by these indicators could better reflect the seedling's quality. The coefficient of determination (R2) between the predicted and measured values based on 1:1 line was 0.95, and the root mean squared error (RMSE) was 0.15, indicating that the model established in this study could precisely reflect the quantitative relationships between the seedling index of S. miltiorrhiza and the environmental factors.

Brassinosteroids (BRs), a group of plant steroidal hormones, play critical roles in many aspects of plant growth and development. Previous studies showed that BRI1-mediated BR signaling regulates cell division and differentiation during Arabidopsis root development via interplaying with auxin and other phytohormones. Arabidopsis somatic embryogenesis receptor-like kinases (SERKs), as co-receptors of BRI1, were found to play a fundamental role in an early activation step of BR signaling pathway. Here we report a novel function of SERKs in regulating Arabidopsis root development. Genetic analyses indicated that SERKs control root growth mainly via a BR-independent pathway. Although BR signaling pathway is completely disrupted in the serk1 bak1 bkk1 triple mutant, the root growth of the triple mutant is much severely damaged than the BR deficiency or signaling null mutants. More detailed analyses indicated that the triple mutant exhibited drastically reduced expression of a number of genes critical to polar auxin transport, cell cycle, endodermis development and root meristem differentiation, which were not observed in null BR biosynthesis mutant cpd and null BR signaling mutant bri1-701.

Arabidopsis contains at least 223 leucine-rich repeat receptor-like protein kinases (LRR-RLKs). The somatic embryogenesis receptor kinases (SERKs) form a small subfamily of the LRR-RLK family. The BRI1-associated receptor kinase (BAK1 or SERK3) was identified as a co-receptor of the brassinosteroid (BR) receptor BRI1. Loss-of-function genetic analysis indicates that BAK1 and its closest paralog BKK1 are independently involved in BRI1-mediated BR signaling and cell-death control signaling pathways. More recently, BAK1 has been identified as a co-receptor interacting with a number of distinct ligand-binding RLKs to regulate multiple signaling pathways. This chapter will mainly discuss how BAK1 and BKK1 were identified as key regulators in controlling cell death, and the possible mechanisms involved in this process.

The Arabidopsis thaliana Somatic Embryogenesis Receptor Kinases (SERKs) consist of five members, SERK1 to SERK5, of the leucine-rich repeat receptor-like kinase subfamily II (LRR-RLK II). SERK3 was named BRI1-Associated Receptor Kinase 1 (BAK1) due to its direct interaction with the brassinosteroid (BR) receptor BRI1 in vivo, while SERK4 has also been designated as BAK1-Like 1 (BKK1) for its functionally redundant role with BAK1. Here we provide genetic and biochemical evidence to demonstrate that SERKs are absolutely required for early steps in BR signaling. Overexpression of four of the five SERKs—SERK1, SERK2, SERK3/BAK1, and SERK4/BKK1—suppressed the phenotypes of an intermediate BRI1 mutant, bri1-5. Overexpression of the kinase-dead versions of these four genes in the bri1-5 background, on the other hand, resulted in typical dominant negative phenotypes, resembling those of null BRI1 mutants. We isolated and generated single, double, triple, and quadruple mutants and analyzed their phenotypes in detail. While the quadruple mutant is embryo-lethal, the serk1 bak1 bkk1 triple null mutant exhibits an extreme de-etiolated phenotype similar to a null bri1 mutant. While overexpression of BRI1 can drastically increase hypocotyl growth of wild-type plants, overexpression of BRI1 does not alter hypocotyl growth of the serk1 bak1 bkk1 triple mutant. Biochemical analysis indicated that the phosphorylation level of BRI1 in serk1 bak1 bkk1 is incapable of sensing exogenously applied BR. As a result, the unphosphorylated level of BES1 has lost its sensitivity to the BR treatment in the triple mutant, indicating that the BR signaling pathway has been completely abolished in the triple mutant. These data clearly demonstrate that SERKs are essential to the early events of BR signaling., Author Summary Brassinosteroids (BRs) are a group of plant hormones critical for plant growth and development. BRs are perceived by a cell-surface receptor complex including two distinctive receptor kinases, BRI1 and BAK1. Whereas BRI1 is a true BR-binding receptor, BAK1 does not appear to have BR-binding activity. Therefore, BAK1 is likely a co-receptor in BR signal transduction. The genetic significance of BAK1 was not clearly demonstrated in previous studies largely due to functional redundancy of BAK1 and its closely related homologues. It was not clear whether BAK1 plays an essential role or only an enhancing role in BR signaling. In this study, we identified all possible BAK1 redundant genes in the Arabidopsis thaliana genome and generated single, double, triple, and quadruple mutants. Detailed analysis indicated that, without BAK1 and its functionally redundant proteins, BR signaling is completely disrupted, largely because BRI1 has lost its ability to activate downstream components. These studies provide the first piece of loss-of-functional genetic evidence that BAK1 is indispensable to the early events of the BR signaling pathway.

Mammal red blood cells (erythrocytes) contain neither nucleus nor mitochondria. Traditional theory suggests that the presence of a nucleus would prevent big nucleated erythrocytes to squeeze through these small capillaries. However, nucleus is too small to hinder erythrocyte deformation. And, there is no sound reason to abandon mitochondria for the living cells. Here, we found that mammal erythrocyte reactive oxygen species (ROS) levels kept stable under diabetes, ischemia reperfusion, and malaria conditions or in vitro sugar/heme treatments, whereas bird erythrocyte ROS levels increased dramatically in these circumstances. Nuclear and mitochondrial extrusion may help mammal erythrocytes to better adapt to high-sugar and high-heme conditions by limiting ROS generation.

Total cellular RNA level is stable usually, although it may increase gradually during growth or decrease gradually under certain stressors. However, we found that mammal cell RNAs could be doubled within 24 h in response to free heme accumulation (ischemia reperfusion and malaria infection) or a high level of glucose treatment (diabetes). Clinical investigations in rats showed that pretreatment with heme (24 h for doubling total RNAs) alleviated oxidative damages caused by diabetes, and pretreatment with glucose (24 h for trebling total RNAs) alleviated oxidative damages caused by ischemia reperfusion or malaria infection. Therefore, this rapid RNA amplification may play an important role in mammal adaptation to diabetes, ischemia reperfusion and malaria infection-derived oxidative stress. This rapid RNA amplification is derived from glucose and heme, but not from their accompanying reactive oxygen species. Hexokinases endure glucose-derived reactive oxygen species accumulation but are not related glucose-derived RNA amplification. In contrast, the TATA box-binding protein (TBP) mediates all glucose- and heme-induced RNA amplification in mammal cells.

In recent years, plant-like proteins in protozoa, metazoa and fungi have been identified. Analysis of them suggests that for millions of years universal plastid endosymbiosis and gene transfer occurred in ancestors of metazoa/fungi, and some transferred fragments have been reserved till now even in modern mammals. Most eukaryotes once contained plastids in the ancient era, and some of them lost plastids later. Functions of homologues in cyanobacterial genomes and eukaryotic genomes are in consensus, and are most involved in organic compound metabolism. With emergence of organelles and subcellular structures in the eukaryotic cell, the locations of these proteins diversified. Furthermore, some novel functions were adopted, especially in vertebrates. Analysis also implies that plastids acquired through a mechanism of secondary endosymbiosis may be preserved even until the multicellular era in simple animals. Phylogenetic trees of some proteins suggest that in ancient times the common ancestor of photosynthetic protist Euglena and parasite Trypanosoma once engulfed a green alga, and then it lost the plastid, but recently some euglenids engulfed algae again. Plastid endosymbiosis is a more general process than we originally thought, and may happen more than one time in one species.

NYB ( Nanchong Yellow Barley ) is a Chl-less barley mutant, which is controlled by a recessive nuclear gene. It is the only protochlorophyllide oxidoreductases (POR)-less barley mutant known in the world. The putative mechanism of the mutation and its Chl synthesis and plastid development are studied here. Neither PORC nor an additional copy of porB could be detected in barley. porB mRNAs are normally expressed and correctly spliced in the mutant. However, the import of PORA, PORB, LHCIIb1 (light harvesting complex II b1) and SSU (small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) proteins to the plastid was greatly hampered in the mutant. We presume that a common translocon is mutated in NYB . The content of the supramolecular light-harvesting POR complex LHPP (light-harvesting NADPH:protochlorophyllide oxidoreductase:protochloroph yllide) and the density of prolamellar bodies in etioplasts are decreased in the mutant. However, no further oxidative damage could be observed for the de-etiolated mutant seedlings after a dark to light shift. Development of the plastid is arrested (less stacking) in NYB , and the mutant becomes more yellowish in high-light conditions, with dwarfi ng of seedlings and decreased yield. The physiological signifi cance and developmental roles of POR proteins and LHPP in barley cells are discussed.

Kinetic studies of protein dephosphorylation in thylakoid membranes showed that the minor light-harvesting antenna protein CP29 could be phosphorylated in barley (C3) and maize (C4) seedlings, but not in spinach under water [Liu, W. J., et al. (2009) Biochim. Biophys. Acta 1787, 1238-1245], salt, or cold stress [Pursiheimo, S., et al. (2003) Plant Cell Environ. 26, 1995-2003], suggesting that phosphorylation of CP29 is a general phenomenon in monocots, but not in dicots under environmental stresses. Abscisic acid (ABA), reactive oxygen species (ROS), salicylic acid (SA), jasmonic acid (JA), ethylene (ET), NO, and the scavenger of H(2)O(2) had weak effects on CP29 phosphorylation. However, three protein kinase inhibitors, U0126, W7, and K252a (for mitogen-activated protein kinase, Ca(2+)-dependent protein kinase, and Ser/Thr protein kinases, respectively), decrease the level of CP29 phosphorylation in barley apparently under environmental stresses. Therefore, these three protein kinases are involved in CP29 phosphorylation. We also found that most CP29 phosphorylation was accompanied by its lateral migration from granum membranes to stroma-exposed thylakoid regions, and the instability of PSII supercomplexes and LHCII trimers under environmental stresses.

Brassinosteroids (BRs) are involved in multiple plant growth and development processes, such as cell elongation, photomorphogenesis, flowering time control, and stress responses. The phytohormone abscisic acid (ABA) is crucial to plant development and adaptation to stressful environments. The receptors and pathways of BRs and ABA have been deeply studied. But the relationship between them remained largely unknown and there are only few reports about it. Our experiments showed that the BR-deficient and BR-insensitive Arabidopsis mutants det2, bri1 - 5 and bri1 - 9 were more sensitive to ABA than the wild type (Ws-2), especially the det2 and bri1 - 9 mutants. Germination, hypocotyl and root elongation, and stomatal apertures of the mutants were more severely inhibited by ABA. All the results suggest that BRs counteract ABA in regulating plant growth, and the interaction may be complicated. The possible mechanisms are discussed.

Previous studies showed that salicylic acid (SA)-deficient transgenic Arabidopsis expressing the salicylate hydroxylase gene NahG had a higher tolerance to moderate salt stress. SA may potentiate the stress response of germination and growth of Arabidopsis seedlings by inducing reactive oxygen species (ROS). However, the detailed mechanism for a better adaption of NahG plants to moderate salt stress is largely unknown. In the present study we found that a higher GSH/GSSG (glutathione/oxidized glutathione) ratio and ASA/DHA (ascorbic acid/dehydroascorbate) ratio in NahG plants during the stress may be the key reason for their stress-tolerance advantage. NahG plants actually could not produce more active antioxidant enzymes than the wild-type ones under natural conditions, but maintain higher activities of glutathione reductase (GR) and dehydroascorbate reductase (DHAR) during the stress. Hereby, the reduced glutathione and reduced ascorbic acid contents are higher in NahG plants under salt stress. However, NahG plants do not adapt better under severe salt stress. All antioxidant enzyme activities, GSH/GSSG ratio and ASA/DHA ratio declined substantively at 400 mM NaCl stress in both NahG and wild-type seedlings.

“NYB” is a chlorophyll-less barley mutant, which grows relatively slow and unhealthily. The effects of water stress on photosystem II (PSII) of NYB and its wild type (WT) were investigated. Unexpected results indicated that the mutant was more resistant to water stress, because: PSII core proteins D1, D2 and LHCII declined more in WT than in NYB under water stress, and the corresponding psbA, psbD and cab mRNAs also decreased more dramatically in WT; CO2 assimilation, stomatal conductance, maximum efficiency of PSII photochemistry (Fv/Fm), efficiency of excitation energy capture by open PSII reaction centres (Fv’/Fm’), quantum yield of PSII electron transport (φPSII) and DCIP photoreduction in NYB were less sensitive to water stress than in WT, although the non-photochemical quenching coefficient (qN) and the photochemical quenching coefficient (qP) were almost the same in NYB and WT. Effective chlorophyll utilization and improved PSII protein formation in the mutant may be the reason for the enhanced stress resistance. Other possible mechanisms are also discussed.

We analyzed nuclear-localized plastid-like DNA (nupDNA) fragments in protozoa, metazoa and fungi. Most eukaryotes that do not have plastids contain 40-5000 bp nupDNAs in their nuclear genomes. These nupDNA fragments are mainly derived from repeated regions of plastids and distribute through the whole genomes. A majority of nupDNA fragments is located on coding regions with very important functions. Similar to plastids, these nupDNAs most possibly originate from cyanobacteria. Analysis of them suggests that through millions of years of universal endosymbiosis and gene transfer they may have occurred in ancient protists before divergence of plants and animals/fungi, and some transferred fragments have been reserved till now even in modern mammals.

Kinetic studies of protein dephosphorylation in barley thylakoid membranes revealed accelerated dephosphorylation of photosystem II (PSII) proteins, and meanwhile rapidly induced phosphorylation of a light-harvesting complex (LHCII) b4, CP29 under water stress. Inhibition of dephosphorylation aggravates stress damages and hampers photosystem recovery after rewatering. This increased dephosphorylation is catalyzed by both intrinsic and extrinsic membrane protein phosphatase. Water stress did not cause any thylakoid destacking, and the lateral migration from granum membranes to stroma-exposed lamellae was only found to CP29, but not other PSII proteins. Activation of plastid proteases and release of TLP40, an inhibitor of the membrane phosphatases, were also enhanced during water stress. Phosphorylation of CP29 may facilitate disassociation of LHCII from PSII complex, disassembly of the LHCII trimer and its subsequent degradation, while general dephosphorylation of PSII proteins may be involved in repair cycle of PSII proteins and stress-response-signaling.