Expression of α-amylases, carbohydrate metabolism, and autophagy in cultured rice cells is coordinately regulated by sugar nutrient

A rice suspension cell culture system has been established to study how sugar depletion regulates α-amylase expression, carbohydrate metabolism, and other physiological and cellular changes. It is shown here that a group of 44 kDa α-amylases are constitutively expressed whether or not the cells are starved of sucrose. However, expression of a new group of α-amylases of 46 kDa is dramatically induced when cells are starved of sucrose. Cellular sugar and starch were rapidly consumed and metabolic activity was decreased in the starved cells. Extensive autophagy also occurred in the starved cells, which caused an increase in vacuolar volume and degradation of cytoplasmic constituents including amyloplasts. Immunocytochemical studies revealed that α-amylases are localized in starch granules within amyloplasts, in cell walls, and in some of the vacuoles. The presence of putative signal sequences in the N-termini of nine rice α-amylases suggests hitherto unidentified pathways for import of α-amylases into amyloplasts. The studies show that differential α-amylase expression, carbohydrate metabolism, metabolic activity, and vacuolar autophagy are coordinately regulated by the sugar level in the medium. As the starved suspension cells exhibit some sugar-regulated characteristics of α-amylase expression in germinating rice embryos as well as physiological changes similar to those in senescing cells, this system represents an ideal tool for studying cellular, biochemical, and molecular biological aspects of α-amylase gene regulation, carbohydrate metabolism, senescence, and protein targeting in plants.     

Microarray-based global differential expression profiling of P450 monooxygenases and regulatory proteins for signal transduction pathways in the white rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Whole genome sequencing of the model white rot basidiomycete Phanerochaete chrysosporium has revealed the largest P450 contingent known to date in fungi, along with related phase I and phase II metabolic genes and signaling cascade genes. As a part of their functional characterization, genome-wide expression profiling under physiologically distinct conditions, nutrient-limited (ligninolytic) and nutrient-rich (non-ligninolytic), was investigated using a custom-designed 70-mer oligonucleotide microarray developed based on 190 target genes and 23 control genes. All 150 P450 genes were found to be expressible under the test conditions, with 27 genes showing differential expression based on a >twofold arbitrary cut-off limit. Of these, 23 P450 genes were upregulated (twofold to ninefold) in defined high-nitrogen cultures whereas four genes were upregulated (twofold to twentyfold) in defined low-nitrogen cultures. Furthermore, tandem P450 member genes in ten of the 16 P450 genomic clusters showed nonassortative regulation of expression reflecting their functional diversity. Full-length cDNAs for two of the high-nitrogen upregulated genes pc-hn1 (CYP5035A1) and pc-hn2 (CYP5036A1) and partial cDNA for a low-nitrogen upregulated gene pc-ln1 (CYP5037A1) were cloned and characterized. The study provided first molecular evidence for the presence of active components of the cAMP- and MAP kinase-signaling pathways in a white rot fungus; four of these components (cpka and ste-12 of cAMP pathway and two MAP kinases, mps1 and sps1) were significantly upregulated (fourfold to eightfold) under nutrient-limited conditions, implying their likely role in the regulation of gene expression involved in secondary metabolism and biodegradation processes under these conditions.     

Quantitative transient gene expression: Comparison of the promoters for maize polyubiquitin1, rice actin1, maize-derivedEmu andCaMV 35S in cells of barley,maize and tobacco

The particle gun approach was used for the quantification of promoter efficiency in a test system for transient gene expression. β-Glucuronidase was used as reporter gene for determining promotote strength. The variability inherent in this gene transfer system was considerably reduced by calculating a transformation efficiency factor given by the expression of a cotransferred second reporter gene (firefly luciferase). The calibration of β-glucuronidase activity by the transformation efficiency factor caused a lower statistical variance of the values and allowed reliable results to be obtained with a smaller set of repetitions. The CaMV 35S promoter (as a control) and the monocot-specific promoters for maize polyubiquitin1, rice actin 1 and the maize-derivedEmu were characterized and compared with respect to expression strength, as tested under identical conditions in suspension cell cultures of maize, barley and tobacco. Compared to the 35S promoter, the monocot-specific promoters show up to 15-fold higher expression in maize and barley but give only weak expression in tobacco. No expression was found for the rice actin 1 promoter in tobacco. The level of reporter gene expression is influenced by the osmotic potential in the agar medium. For theEmu promoter, the calibrated β-glucuronidase activities remained mearly constant at low sucrose concentrations. Above 8% sucrose, the calibrated activities increased steadily with increasing osmotic conditions, reaching a three-to four-fold higher level at the highest sucrose concentration (32%) as compared to the standard concentration (4% sucrose) in the medium.     

Sugar starvation- and GA-inducible calcium-dependent protein kinase 1 feedback regulates GA biosynthesis and activates a 14-3-3 protein to confer drought tolerance in rice seedlings

Germination followed by seedling growth constitutes two essential steps in the initiation of a new life cycle in plants, and in cereals, completion of these steps is regulated by sugar starvation and the hormone gibberellin. A calcium-dependent protein kinase 1 gene (OsCDPK1) was identified by differential screening of a cDNA library derived from sucrose-starved rice suspension cells. The expression of OsCDPK1 was found to be specifically activated by sucrose starvation among several stress conditions tested as well as activated transiently during post-germination seedling growth. In gain- and loss-of-function studies performed with transgenic rice overexpressing a constitutively active or RNA interference gene knockdown construct, respectively, OsCDPK1 was found to negatively regulate the expression of enzymes essential for GA biosynthesis. In contrast, OsCDPK1 activated the expression of a 14-3-3 protein, GF14c. Overexpression of either constitutively active OsCDPK1 or GF14c enhanced drought tolerance in transgenic rice seedlings. Hence, our studies demonstrated that OsCDPK1 transduces the post-germination Ca²⁺ signal derived from sugar starvation and GA, refines the endogenous GA concentration and prevents drought stress injury, all essential functions to seedling development at the beginning of the life cycle in rice.     

Expression profiling of <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> metabolic genes using DNA microarray analysis

Doxorubicin (DXR) and daunorubicin (DNR) are anthracycline antibiotics produced by Streptomyces peucetius and widely used as cancer chemotherapeutic agents. To improve their productivity, regulation of DXR/DNR synthesis genes as well as central metabolic pathway genes must be understood more clearly. So far, studies have focused on DXR/DNR gene regulation. To investigate the correlation between the central metabolic pathway genes and DXR/DNR productivity, we selected 265 genes involved in glycolysis, fermentation, the citric acid cycle, butanoate metabolism, etc., and searched for their sequences in the S. peucetius genome by comparing gene sequences to those of Streptomyces coelicolor. The homologous genes were amplified by PCR and arrayed on glass microarray slides. Gene expression was monitored under two different growth media conditions, R2YE and NDYE. Genes involved in the production of malonyl-CoA and propionyl-CoA, the main precursors for doxorubicin synthesis, were mainly upregulated in NDYE media. Genes related to acetyl-CoA and the urea cycle were also upregulated. These changes in gene expression were confirmed by real-time RT-PCR.     

Metabolic derepression of alpha-amylase gene expression in suspension-cultured cells of rice

We present evidence to show that the alpha-amylase gene family in rice is under two different modes of regulation: 1) hormonal regulation in germinating seeds, and 2) metabolic repression in cultured cells by available carbohydrate nutrients. Expression of alpha-amylase genes in deembryoed rice seeds is known to be induced by exogenous gibberellic acid. On the other hand, expression of alpha-amylase genes in suspension-cultured cells is induced by the deprivation of carbohydrate nutrient. A lag period of 2-4 h is required for the induction of alpha-amylase mRNA in sucrose-depleted medium. The induction of alpha-amylase expression is extraordinarily high and levels of alpha-amylase mRNA can be increased 8-20-folds after 24 h of sucrose starvation. The synthesis and secretion of alpha-amylase is also dependent upon the level of carbon source. The derepression or repression of alpha-amylase synthesis can be readily reversed by the deprivation or replenishment of sucrose in the medium, respectively. Glucose and fructose exert a repression on the alpha-amylase synthesis similar to that of sucrose. A hypothesis that explains the induction of alpha-amylase synthesis by carbohydrate starvation is proposed. Our data have suggested a hitherto undiscovered, potentially important control mechanism of carbohydrate metabolism in higher plants.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> RETINOBLASTOMA-RELATED PROTEIN 1 is involved in G1 phase cell cycle arrest caused by sucrose starvation

Although sucrose availability is crucial for commitment to plant cell division during G1 phase by controlling the expression of D-type cyclins, it has remained unclear how these factors mediate entry into the cell cycle. Here we show that Arabidopsis RETINOBLASTOMA-RELATED PROTEIN 1 (AtRBR1) is involved in G1-phase cell cycle arrest caused by sucrose starvation. We generated estrogen-inducible AtRBR1 RNA interference (RNAi) Arabidopsis suspension MM2d cells, and found that downregulation of AtRBR1 leads to a higher frequency of arrest in G2 phase, instead of G1-phase arrest in the uninduced control, after sucrose starvation. Synchronization experiments confirmed that downregulation of AtRBR1 leads to a prolonged G2 phase and delayed activation of G2/M marker genes. Downregulation of AtRBR1 also stimulated the activation of E2F-regulated genes when these genes were repressed in the uninduced cells under the limited sucrose conditions. We conclude that AtRBR1 is a key effector for the ability of sucrose to modulate progression from G1 phase.     

Lanthanide ions are agonists of transient gene expression in rice protoplasts and act in synergy with ABA to increase Em gene expression

Previous work has shown that in rice suspension cells, NaCl at 0.4 M can induce Em gene expression and act synergistically with ABA, possibly by potentiating the ABA response pathway through a rate-limiting intermediate (R.M. Bostock and R.S. Quatrano (1992) Plant Physiol., 98, 1356–1363). Since calcium is an intermediate in ABA regulation of stomatal closure, we tested the effect of calcium changes on ABA-inducible Em gene expression in transiently transformed rice protoplasts. We show that calcium is required for ABA-inducible Em-GUS expression and can act in synergy with ABA. The trivalent ions lanthanum, gadolinium, and aluminum, which are known to interact with calcium- and other signaling pathways, can act at sub-millimolar concentrations to increase GUS reporter gene expression driven by several promoters in transiently transformed rice protoplasts. This effect is not specific for the ABA-inducible Em promoter, but is synergistic with ABA. The lanthanum synergy with ABA does not require calcium. In rice suspension cells, lanthanum alone does not induce Em gene expression, in contrast to transiently transformed protoplasts, yet can act synergistically with ABA to effectively increase the sensitivity to ABA greater than tenfold. Trivalent ions may be a useful tool to study the regulation of gene expression. The possible effects of trivalent ions on ABA signal transduction and gene expression are discussed.     

水稻条斑病细菌hrp基因诱导表达系统的建立

水稻条斑病细菌(Xanthomonas oryzae pv.oryzicola,Xooc)决定在非寄主植物上激发过敏反应(hypersensitive response)和在寄主水稻上具致病性(pathogenicity)的hrp基因簇是诱导表达的。为研究hrp基因的功能,利用hpa1和hrpX基因的启动子与gfp基因进行融合,构建了hrp基因诱导表达系统。绿色荧光蛋白表达揭示,Xooc的hrp基因在营养丰富的NB培养基上不能有效表达,在hrp诱导培养基XOM3上可有效表达。以hrpX和hrpG突变体为参照,RT-PCR研究结果提示,Xooc野生型菌株hpa1基因在NB上不能有效表达,在XOM3培养基上可有效表达。相应地,hrpX突变体中hpa1基因不能被诱导表达,而在hrpG突变体中hpa1基因转录表达水平低于野生菌。研究结果还证实,水稻悬浮细胞能高效诱导Xooc的hrp基因表达。Xooc hrp基因诱导表达系统的建立为研究hrp基因功能、发掘T3SS效应分子以及开展Xooc致病性研究奠定了基础。     

Production of functional recombinant bovine trypsin in transgenic rice cell suspension cultures

A synthetic bovine trypsinogen (sbTrypsinogen) was synthesized on the basis of rice-optimized codon usage via an overlap PCR strategy, prior to being expressed under the control of the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Secretion of trypsin into the culture medium was achieved by using the existing signal peptide. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), mediated by Agrobacterium tumefaciens. The integration of the sbTrypsinogen gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification, and sbTrypsin expression in transgenic rice suspension cells was confirmed via Northern blot analysis. Western blot analysis detected glycosylated proteins in the culture medium, having masses from 24 to 26 kDa, following induction by sugar starvation. Proteolytic activity of the rice-derived trypsin was confirmed by gelatin zymogram, and was similar to that of the commercial bovine-produced trypsin. The yields of sbTrypsin that accumulated in the transgenic rice cell suspension medium were 15 mg/L at 5 days after sugar starvation.