OsbZIP38/87-mediated activation of OsHXK7 improves the viability of rice cells under hypoxic conditions.

Maintenance of energy metabolism is critical for rice (Oryza sativa) tolerance under submerged cultivation. Here, OsHXK7 was the most actively induced hexokinase gene in the embryos of hypoxically germinating rice seeds. Suspension-cultured cells established from seeds of T-DNA null mutants for the OsHXK7 locus did not regrow after 3-d-hypoxic stress and showed increased susceptibility to low-oxygen stress-in terms of viability-and decreased alcoholic fermentation activities compared to those of the wild-type. The promoter element containing the TGACG-motif, a well-known target site for the basic leucine zipper (bZIP) transcription factors, was responsible for sugar regulation of the OsHXK7 promoter activity. Systematic screening of the OsbZIP genes showing the similar expression patterns to that of OsHXK7 in the transcriptomic datasets produced two bZIP genes, OsbZIP38 and 87, belonging to the S1 bZIP subfamily as the candidate for the activator for this gene expression. Gain- and loss-of-function experiments through transient expression assays have demonstrated that these two bZIP proteins are indeed involved in the induction of OsHXK7 expression under starvation or low-energy conditions. Our finding suggests that C/S1 bZIP network-mediated hypoxic deregulation of sugar-responsive genes may work in concert for the molecular adaptation of rice cells to submergence.

A rice seed-specific glycine-rich protein OsDOR1 interacts with GID1 to repress GA signaling and regulates seed dormancy.

Seed dormancy is an important agronomic trait under the control of complex genetic and environmental interactions, which have not been yet comprehensively understood. From the field screening of rice mutant library generated by a Ds transposable element, we identified a pre-harvest sprouting (PHS) mutant dor1. This mutant has a single insertion of Ds element at the second exon of OsDOR1 (LOC_Os03g20770), which encodes a novel seed-specific glycine-rich protein. This gene successfully complemented the PHS phenotype of dor1 mutant and its ectopic expression enhanced seed dormancy. Here, we demonstrated that OsDOR1 protein binds to the GA receptor protein, OsGID1 in rice protoplasts, and interrupts with the formation OsGID1-OsSLR1 complex in yeast cells. Co-expression of OsDOR1 with OsGID1 in rice protoplasts attenuated the GA-dependent degradation of OsSLR1, the key repressor of GA signaling. We showed the endogenous OsSLR1 protein level in the dor1 mutant seeds is significantly lower than that of wild type. The dor1 mutant featured a hypersensitive GA-response of α-amylase gene expression during seed germination. Based on these findings, we suggest that OsDOR1 is a novel negative player of GA signaling operated in the maintenance of seed dormancy. Our findings provide a novel source of PHS resistance.

Abscisic acid activation of oleosin gene HvOle3 expression prevents the coalescence of protein storage vacuoles in barley aleurone cells.

Protein storage vacuoles (PSVs) in aleurone cells coalesce during germination, and this process is highly coupled with mobilization of PSV reserves, allowing de novo synthesis of various hydrolases in aleurone cells for endosperm degradation. Here we show that in barley (Hordeum vulgare L.) oleosins, the major integral proteins of oleosomes are encoded by four genes (HvOle1 to 4), and the expression of HvOle1 and HvOle3 is strongly up-regulated by abscisic acid (ABA), which shows antagonism to gibberellic acid. In aleurone cells, all HvOLEs were subcellularly targeted to the tonoplast of PSVs. Gain-of-function analyses revealed that HvOLE3 effectively delayed PSV coalescence, whereas HvOLE1 only had a moderate effect, with no notable effect of HvOLE2 and 4. With regard to longevity, HvOLE3 chiefly outperformed other HvOLEs, followed by HvOLE1. Experiments swapping the N- and C-terminal domain between HvOLE3 and other HvOLEs showed that the N-terminal region of HvOLE3 is mainly responsible, with some positive effect by the C-terminal region, for mediating the specific preventive effect of HvOLE3 on PSV coalescence. Three ACGT-core elements and the RY-motif were responsible for ABA induction of HvOle3 promoter activity. Transient expression assays using aleurone protoplasts demonstrated that transcriptional activation of the HvOle3 promoter was mediated by transcription factors HvABI3 and HvABI5, which acted downstream of protein kinase HvPKABA1.

Comparison of transcriptomic adjustments to availability of sugar, cellular energy, and oxygen in germinating rice embryos.

During germination, the availability of sugars, oxygen, or cellular energy fluctuates under dynamic environmental conditions, likely affecting the global RNA profile of rice genes. Most genes that exhibit sugar-regulation in rice embryos under aerobic conditions are responsive to low energy and anaerobic conditions, indicating that sugar regulation is strongly associated with energy and anaerobic signaling. The interference pattern of sugar regulation by either anaerobic or low energy conditions indicates that induction is likely the more prevalent regulatory mechanism than repression for altering the expression of sugar-regulated genes. Among the aerobically sugar-regulated genes, limited genes exhibit sugar regulation under anaerobic conditions, indicating that anaerobic conditions strongly influence sugar regulated gene expression. Anaerobically responsive genes substantially overlap with low energy responsive genes. In particular, the expression levels of anaerobically downregulated genes are consistent with those provoked by low energy conditions, suggesting that anaerobic downregulation results from the prevention of aerobic respiration due to the absence of the final electron acceptor, i.e., molecular oxygen. It has been noted that abscisic acid (ABA) responsive genes are over representative of genes upregulated under low energy conditions, in contrast to downregulated genes. This suggests that either ABA itself or upstream signaling components of the ABA signaling pathway are likely to be involved in the signaling pathways activated by low energy conditions.

Aquaporin activity of barley tonoplast intrinsic proteins is involved in the delay of the coalescence of protein storage vacuoles in aleurone cells.

The coalescence of protein storage vacuoles (PSVs) is one of the most prominent cellular changes occurring in cereal aleurone cells during germination. This structural change is highly coupled with the functional transition of this organelle from a storage compartment to a lytic section. Gibberellic acid (GA) promotes this process, whereas abscisic acid (ABA) prevents it. Previously, we demonstrated that ABA-inducible HvTIP3;1 plays a decisive role in ABA-mediated prevention of PSV fusion. In this follow-up study, we examined whether the aquaporin activity of tonoplast intrinsic protein (TIP) is related to its preventive effect on PSV fusion using various functional mutants. The defective forms of aquaporin (HvTIP3;1m and HvTIP3;1ΔNPA-GFPs for HvTIP3;1, and HvTIP1;2m for HvTIP1;2) were found to be less effective than the usual form in delaying the PSV fusion process occurring in GA-treated cells. In contrast, overexpression of HvTIP3;1m reduced the preventive effect of ABA on PSV fusion. Upon inhibition of aquaporin activity using mercury, PSV fusion occurred to a greater extent in ABA-treated barley protoplasts. These data suggest that the aquaporin activity of TIP is involved in the deterrent effect of TIP on PSV coalescence. TIP3-GFP barley transgenic seeds showed prolonged expression of the TIP3;1 transcript. Moreover, PSV fusion progressed at a much slower rate compared to wild type. Additionally, the degradation of storage proteins was not as efficient, suggesting that a metamorphic transition of PSVs to lytic organelles is closely correlated with the disappearance of HvTIPs and the PSV fusion process.

Cross-talk between ABA and sugar signaling is mediated by the ACGT core and CE1 element reciprocally in OsTIP3;1 promoter.

Recently, much effort has been made to determine the molecular links and cross-talk between sugar and abscisic acid (ABA) signaling pathways. ABA-inducible expression of OsTIP3;1, encoding a rice tonoplast intrinsic protein, was enhanced by sugar depletion. Such a stimulatory increase in OsTIP3;1 expression under sugar-starvation is possibly not owing to changes in endogenous ABA content. The transient expression assay indicated that the 5' flanking region of OsTIP3;1 delivered similar collaborative responsiveness to starvation and ABA, suggesting that this gene promoter could be a good molecular probe to examine the interaction between sugar and ABA signaling pathways. Targeted mutagenesis demonstrated that disruption of ACGT cores decreased the induction of OsTIP3;1 promoter activity under either starvation or ABA, whereas mutation of coupling element 1 (CE1), which is an ABI4 binding site, reversely increased it, suggesting that those two distinct cis-regulatory elements reciprocally regulate the responsiveness of this promoter to both sugar and ABA. Consistent with this result, antisense inhibition of ABI4 increased the OsTIP3;1 promoter activity. ABI4 expression was also enhanced by sugars and repressed by ABA, suggesting that reduced ABI4 binding to CE1 in the absence of sugar and presence of ABA could increase ABA-induction of the OsTIP3;1 promoter activity.

Dissection of cis-regulatory element architecture of the rice oleosin gene promoters to assess abscisic acid responsiveness in suspension-cultured rice cells.

Oleosins are the most abundant proteins in the monolipid layer surrounding neutral storage lipids that form oil bodies in plants. Several lines of evidence indicate that they are physiologically important for the maintenance of oil body structure and for mobilization of the lipids stored inside. Rice has six oleosin genes in its genome, the expression of all of which was found to be responsive to abscisic acid (ABA) in our examination of mature embryo and aleurone tissues. The 5'-flanking region of OsOle5 was initially characterized for its responsiveness to ABA through a transient expression assay system using the protoplasts from suspension-cultured rice cells. A series of successive deletions and site-directed mutations identified five regions critical for the hormonal induction of its promoter activity. A search for cis-acting elements in these regions deposited in a public database revealed that they contain various promoter elements previously reported to be involved in the ABA response of various genes. A gain-of-function experiment indicated that multiple copies of all five regions were sufficient to provide the minimal promoter with a distinct ABA responsiveness. Comparative sequence analysis of the short, but still ABA-responsive, promoters of OsOle genes revealed no common modular architecture shared by them, indicating that various distinct promoter elements and independent trans-acting factors are involved in the ABA responsiveness of rice oleosin multigenes.

Interplay between ABA and GA Modulates the Timing of Asymmetric Cell Divisions in the Arabidopsis Root Ground Tissue.

In multicellular organisms, controlling the timing and extent of asymmetric cell divisions (ACDs) is crucial for correct patterning. During post-embryonic root development in Arabidopsis thaliana, ground tissue (GT) maturation involves an additional ACD of the endodermis, which generates two different tissues: the endodermis (inner) and the middle cortex (outer). It has been reported that the abscisic acid (ABA) and gibberellin (GA) pathways are involved in middle cortex (MC) formation. However, the molecular mechanisms underlying the interaction between ABA and GA during GT maturation remain largely unknown. Through transcriptome analyses, we identified a previously uncharacterized C2H2-type zinc finger gene, whose expression is regulated by GA and ABA, thus named GAZ (GA- AND ABA-RESPONSIVE ZINC FINGER). Seedlings ectopically overexpressing GAZ (GAZ-OX) were sensitive to ABA and GA during MC formation, whereas GAZ-SRDX and RNAi seedlings displayed opposite phenotypes. In addition, our results indicated that GAZ was involved in the transcriptional regulation of ABA and GA homeostasis. In agreement with previous studies that ABA and GA coordinate to control the timing of MC formation, we also confirmed the unique interplay between ABA and GA and identified factors and regulatory networks bridging the two hormone pathways during GT maturation of the Arabidopsis root.

Role of rice cytosolic hexokinase OsHXK7 in sugar signaling and metabolism.

We characterized the function of the rice cytosolic hexokinase OsHXK7 (Oryza sativa Hexokinase7), which is highly upregulated when seeds germinate under O2 -deficient conditions. According to transient expression assays that used the promoter:luciferase fusion construct, OsHXK7 enhanced the glucose (Glc)-dependent repression of a rice α-amylase gene (RAmy3D) in the mesophyll protoplasts of maize, but its catalytically inactive mutant alleles did not. Consistently, the expression of OsHXK7, but not its catalytically inactive alleles, complemented the Arabidopsis glucose insensitive2-1 (gin2-1) mutant, thereby resulting in the wild type characteristics of Glc-dependent repression, seedling development, and plant growth. Interestingly, OsHXK7-mediated Glc-dependent repression was abolished in the O2 -deficient mesophyll protoplasts of maize. This result provides compelling evidence that OsHXK7 functions in sugar signaling via a glycolysis-dependent manner under normal conditions, but its signaling role is suppressed when O2 is deficient. The germination of two null OsHXK7 mutants, oshxk7-1 and oshxk7-2, was affected by O2 deficiency, but overexpression enhanced germination in rice. This result suggests the distinct role that OsHXK7 plays in sugar metabolism and efficient germination by enforcing glycolysis-mediated fermentation in O2 -deficient rice.

The Arabidopsis thaliana NGATHA transcription factors negatively regulate cell proliferation of lateral organs.

The cell proliferation process of aerial lateral organs, such as leaves and flowers, is coordinated by complex genetic networks that, in general, converge on the cell cycle. The Arabidopsis thaliana NGATHA (AtNGA) family comprises four members that belong to the B3-type transcription factor superfamily, and has been suggested to be involved in growth and development of aerial lateral organs, although its role in the cell proliferation and expansion processes remains to be resolved in more detail. In order to clarify the role of AtNGAs in lateral organ growth, we took a systematic approach using both the loss- and gain-of-functional mutants of all four members. Our results showed that overexpressors of AtNGA1 to AtNGA4 developed small, narrow lateral organs, whereas the nga1 nga2 nga3 nga4 quadruple mutant produced large, wide lateral organs. We found that cell numbers of the lateral organs were significantly affected: a decrease in overexpressors and, inversely, an increase in the quadruple mutant. Kinematic analyses on leaf growth revealed that, compared with the wild type, the overexpressors displayed a lower activity of cell proliferation and yet the mutant a higher activity. Changes in expression of cell cycle-regulating genes were well in accordance with the cell proliferation activities, establishing that the AtNGA transcription factors act as bona fide negative regulators of the cell proliferation of aerial lateral organs.

Brassinazole resistant 1 (BZR1)-dependent brassinosteroid signalling pathway leads to ectopic activation of quiescent cell division and suppresses columella stem cell differentiation.

Previous publications have shown that BRI1 EMS suppressor 1 (BES1), a positive regulator of the brassinosteroid (BR) signalling pathway, enhances cell divisions in the quiescent centre (QC) and stimulates columella stem cell differentiation. Here, it is demonstrated that BZR1, a BES1 homologue, also promotes cell divisions in the QC, but it suppresses columella stem cell differentiation, opposite to the action of BES1. In addition, BR and its BZR1-mediated signalling pathway are shown to alter the expression/subcellular distribution of pin-formed (PINs), which may result in changes in auxin movement. BR promotes intense nuclear accumulation of BZR1 in the root tip area, and the binding of BZR1 to the promoters of several root development-regulating genes, modulating their expression in the root stem cell niche area. These BZR1-mediated signalling cascades may account for both the ectopic activation of QC cell divisions as well as the suppression of the columella stem cell differentiation. They could also inhibit auxin-dependent distal stem cell differentiation by antagonizing the auxin/WOX5-dependent pathway. In conclusion, BZR1-/BES1-mediated BR signalling pathways show differential effects on the maintenance of root apical meristem activities: they stimulate ectopic QC division while they show opposite effects on the differentiation of distal columella stem cells in a BR concentration- and BZR1-/BES1-dependent manner.

Ratio of phosphorylated HSP27 to nonphosphorylated HSP27 biphasically acts as a determinant of cellular fate in gemcitabine-resistant pancreatic cancer cells.

Gemcitabine has been used most commonly as an anticancer drug to treat advanced pancreatic cancer patients. However, intrinsic or acquired resistance of pancreatic cancer to gemcitabine was also developed, which leads to very low five-year survival rates. Here, we investigated whether cellular levels of HSP27 phosphorylation act as a determinant of cellular fate with gemcitabine. In addition we have demonstrated whether HSP27 downregulation effectively could overcome the acquisition of gemcitabine resistance by using transcriptomic analysis. We observed that gemcitabine induced p38/HSP27 phosphorylation and caused acquired resistance. After acquisition of gemcitabine resistance, cancer cells showed higher activity of NF-κB. NF-κB activity, as well as colony formation in gemcitabine-resistant pancreatic cancer cells, was significantly decreased by HSP27 downregulation and subsequent TRAIL treatment, showing that HSP27 was a common network mediator of gemcitabine/TRAIL-induced cell death. After transcriptomic analysis, gene fluctuation after HSP27 downregulation was very similar to that of pancreatic cancer cells susceptible to gemcitabine, and then in opposite position to that of acquired gemcitabine resistance, which makes it possible to downregulate HSP27 to overcome the acquired gemcitabine resistance to function as an overall survival network inhibitor. Most importantly, we demonstrated that the ratio of phosphorylated HSP27 to nonphosphorylated HSP27 rather than the cellular level of HSP27 itself acts biphasically as a determinant of cellular fate in gemcitabine-resistant pancreatic cancer cells.

Abscisic acid prevents the coalescence of protein storage vacuoles by upregulating expression of a tonoplast intrinsic protein gene in barley aleurone.

Tonoplast intrinsic proteins (TIPs) are integral membrane proteins that are known to function in plants as aquaporins. Here, we propose another role for TIPs during the fusion of protein storage vacuoles (PSVs) in aleurone cells, a process that is promoted by gibberellic acid (GA) and prevented by abscisic acid (ABA). Studies of the expression of barley (Hordeum vulgare) TIP genes (HvTIP) showed that GA specifically decreased the abundance of HvTIP1;2 and HvTIP3;1 transcripts, while ABA strongly increased expression of HvTIP3;1. Increased or decreased expression of HvTIP3;1 interfered with the hormonal effects on vacuolation in aleurone protoplasts. HvTIP3;1 gain-of-function experiments delayed GA-induced vacuolation, whereas HvTIP3;1 loss-of-function experiments promoted vacuolation in ABA-treated aleurone cells. These results indicate that TIP plays a key role in preventing the coalescence of small PSVs in aleurone cells. Hormonal regulation of the HvTIP3;1 promoter is similar to the regulation of the endogenous gene, indicating that induction of the transcription of HvTIP3;1 by ABA is a critical factor in the prevention of PSV coalescence in response to ABA. Promoter analysis using deletions and site-directed mutagenesis of sequences identified three cis-acting elements that are responsible for ABA responsiveness in the HvTIP3;1 promoter. Promoter analysis also showed that ABA responsiveness of the HvTIP3;1 promoter is likely to occur via a unique regulatory system distinct from that involving the ABA-response promoter complexes.

Comparative analyses of lipidomes and transcriptomes reveal a concerted action of multiple defensive systems against photooxidative stress in Haematococcus pluvialis.

Haematococcus pluvialis cells predominantly remain in the macrozooid stage under favourable environmental conditions but are rapidly differentiated into haematocysts upon exposure to various environmental stresses. Haematocysts are characterized by massive accumulations of astaxanthin sequestered in cytosolic oil globules. Lipidomic analyses revealed that synthesis of the storage lipid triacylglycerol (TAG) was substantially stimulated under high irradiance. Simultaneously, remodelling of membrane glycerolipids occurred as a result of dramatic reductions in chloroplast membrane glycolipids but remained unchanged or declined slightly in extraplastidic membrane glycerolipids. De novo assembly of transcriptomes revealed the genomic and metabolic features of this unsequenced microalga. Comparative transcriptomic analysis showed that so-called resting cells (haematocysts) may be more active than fast-growing vegetative cells (macrozooids) regarding metabolic pathways and functions. Comparative transcriptomic analyses of astaxanthin biosynthesis suggested that the non-mevalonate pathway mediated the synthesis of isopentenyl diphosphate, as the majority of genes involved in subsequent astaxanthin biosynthesis were substantially up-regulated under high irradiance, with the genes encoding phytoene synthase, phytoene desaturase, and ß-carotene hydroxylase identified as the most prominent regulatory components. Accumulation of TAG under high irradiance was attributed to moderate up-regulation of de novo fatty acid biosynthesis at the gene level as well as to moderate elevation of the TAG assembly pathways. Additionally, inferred from transcriptomic differentiation, an increase in reactive oxygen species (ROS) scavenging activity, a decrease in ROS production, and the relaxation of over-reduction of the photosynthetic electron transport chain will work together to protect against photooxidative stress in H. pluvialis under high irradiance.

The Arabidopsis thaliana GRF-INTERACTING FACTOR gene family plays an essential role in control of male and female reproductive development.

Reproductive success of angiosperms relies on the precise development of the gynoecium and the anther, because their primary function is to bear and to nurture the embryo sac/female gametophyte and pollen, in which the egg and sperm cells, respectively, are generated. It has been known that the GRF-INTERACTING FACTOR (GIF) transcription co-activator family of Arabidopsis thaliana (Arabidopsis) consists of three members and acts as a positive regulator of cell proliferation. Here, we demonstrate that GIF proteins also play an essential role in development of reproductive organs and generation of the gamete cells. The gif1 gif2 gif3 triple mutant, but not the single or double mutants, failed to establish normal carpel margin meristem (CMM) and its derivative tissues, such as the ovule and the septum, resulting in a split gynoecium and no observable embryo sac. The gif triple mutant also displayed severe structural and functional defects in the anther, producing neither microsporangium nor pollen grains. Therefore, we propose that the GIF family of Arabidopsis is a novel and essential component required for the cell specification maintenance during reproductive organ development and, ultimately, for the reproductive competence.

Differential anoxic expression of sugar-regulated genes reveals diverse interactions between sugar and anaerobic signaling systems in rice.

The interaction between the dual roles of sugar as a metabolic fuel and a regulatory molecule was unveiled by examining the changes in sugar signaling upon oxygen deprivation, which causes the drastic alteration in the cellular energy status. In our study, the expression of anaerobically induced genes is commonly responsive to sugar, either under the control of hexokinase or non-hexokinase mediated signaling cascades. Only sugar regulation via the hexokinase pathway was susceptible for O2 deficiency or energy deficit conditions evoked by uncoupler. Examination of sugar regulation of those genes under anaerobic conditions revealed the presence of multiple paths underlying anaerobic induction of gene expression in rice, subgrouped into three distinct types. The first of these, which was found in type-1 genes, involved neither sugar regulation nor additional anaerobic induction under anoxia, indicating that anoxic induction is a simple result from the release of sugar repression by O2-deficient conditions. In contrast, type-2 genes also showed no sugar regulation, albeit with enhanced expression under anoxia. Lastly, expression of type-3 genes is highly enhanced with sugar regulation sustained under anoxia. Intriguingly, the inhibition of the mitochondrial ATP synthesis can reproduce expression pattern of a specific set of anaerobically induced genes, implying that rice cells may sense O2 deprivation, partly via perception of the perturbed cellular energy status. Our study of interaction between sugar signaling and anaerobic conditions has revealed that sugar signaling and the cellular energy status are likely to communicate with each other and influence anaerobic induction of gene expression in rice.

Comparative transcriptome profiling of developing caryopses from two rice cultivars with differential dormancy.

Pre-harvest sprouting (PHS) in rice causes poor grain quality and results in significant reductions in yield, leading to significant economic losses. In contrast, deep dormancy can lead to equally unwanted non-uniform germination. Therefore, a suitable level of dormancy is a critically important agronomic trait. In this study, an analysis of PHS in developing seeds of two Korean rice cultivars (vivipary), Gopum and Samgwang, revealed differences in dormancy in caryopses at 25 d after heading (DAH). To assess the transcriptomic characteristics associated with vivipary, we compared RNA profiles at early (3-6 DAH), middle (25 DAH), and late (40 DAH) developmental stages. Transcriptomic differentiation was most pronounced in caryopses at 25 DAH, the developmental stage at which differential dormancy was also the most prominent. A k-means clustering analysis of the two cultivars revealed groups of genes with similar or dissimilar expression profiles. Many of the genes that showed distinct differential expression profiles were those involved in seed maturation. Intriguingly, differential gene expression levels between the two cultivars were positively correlated with fold-changes in their expression during the early half of caryopsis development. This implies that the establishment of seed dormancy is strongly correlated with the altered transcriptomic patterns related to the progression of maturation. Our global RNA profiling suggests that caryopsis development in Gopum proceeds at a greater speed than in the Samgwang cultivar. Thus, a high degree of maturity and early dormancy release may be present in 25 DAH caryopses of Gopum, although we cannot exclude the possibility of genetic defects modifying dormancy. The comparative transcriptomic analysis of the two cultivars did not reveal noticeable differences in RNA profiles with respect to differences in abscisic acid (ABA) content or ABA sensitivity. Therefore, it is unlikely that ABA is directly involved in the differences in dormancy observed between the two cultivars.

Hexokinase-mediated sugar signaling controls expression of the calcineurin B-like interacting protein kinase 15 gene and is perturbed by oxidative phosphorylation inhibition.

Calcineurin B-like (CBL) interacting protein kinase 15 (CIPK15) is a newly identified positive regulator which is critical to directing the O(2) deficiency signal to the sugar signaling cascade as part of Amy3D (representative Amy3 gene) regulation in rice. It is located upstream and probably contributes to reserve mobilization under anoxia. In isolated starving embryos, the temporal pattern of accumulation of CIPK15 transcripts and leaky suppression of this gene suggests that factors other than CIPK15 may also be involved in the regulation of Amy3D expression. Probing of a variety of sugars and sugar analogs has shown that hexokinase mediates the sugar regulation of CIPK15. For example, hexokinase substrates, such as mannose, 2-deoxyglucose, and other metabolizable sugars, repressed CIPK15 expression, whereas 3-O-methylglucose and 6-deoxyglucose did not. By using glucosamine, a hexokinase inhibitor, to release glucose-dependent CIPK15 suppression, we confirmed that hexokinase mediates regulation of this gene. Chemical inhibitors of mitochondrial electron transfer, proton separation or ATP synthase also effectively abolished sugar-induced repression of CIPK15. This type of interference, the release from glucose-induced repression of gene expression by inhibition of oxidative phosphorylation, was previously identified for the Amy3D gene, which suggests that hexokinase-mediated sugar signaling may be coordinated with the cellular energy status. Analysis of a transgenic rice cell line harboring the GUS reporter gene under the control of the CIPK15 promoter, and transient expression assay for 3' UTR of the CIPK15 gene indicate that sugar regulation of the rice CIPK15 gene is likely mediated by 2548-bp 5'-flanking region, with no additional post-transcriptional control.

Analysis of Arabidopsis glucose insensitive growth mutants reveals the involvement of the plastidial copper transporter PAA1 in glucose-induced intracellular signaling.

Sugars play important roles in many aspects of plant growth and development, acting as both energy sources and signaling molecules. With the successful use of genetic approaches, the molecular components involved in sugar signaling have been identified and their regulatory roles in the pathways have been elucidated. Here, we describe novel mutants of Arabidopsis (Arabidopsis thaliana), named glucose insensitive growth (gig), identified by their insensitivity to high-glucose (Glc)-induced growth inhibition. The gig mutant displayed retarded growth under normal growth conditions and also showed alterations in the expression of Glc-responsive genes under high-Glc conditions. Our molecular identification reveals that GIG encodes the plastidial copper (Cu) transporter PAA1 (for P(1B)-type ATPase 1). Interestingly, double mutant analysis indicated that in high Glc, gig is epistatic to both hexokinase1 (hxk1) and aba insensitive4 (abi4), major regulators in sugar and retrograde signaling. Under high-Glc conditions, the addition of Cu had no effect on the recovery of gig/paa1 to the wild type, whereas exogenous Cu feeding could suppress its phenotype under normal growth conditions. The expression of GIG/PAA1 was also altered by mutations in the nuclear factors HXK1, ABI3, and ABI4 in high Glc. Furthermore, a transient expression assay revealed the interaction between ABI4 and the GIG/PAA1 promoter, suggesting that ABI4 actively regulates the transcription of GIG/PAA1, likely binding to the CCAC/ACGT core element of the GIG/PAA1 promoter. Our findings indicate that the plastidial Cu transporter PAA1, which is essential for plastid function and/or activity, plays an important role in bidirectional communication between the plastid and the nucleus in high Glc.

Interference with oxidative phosphorylation enhances anoxic expression of rice alpha-amylase genes through abolishing sugar regulation.

Rice has the unique ability to express alpha-amylase under anoxic conditions, a feature that is critical for successful anaerobic germination and growth. Previously, anaerobic conditions were shown to up-regulate the expression of Amy3 subfamily genes (Amy3B/C, 3D, and 3E) in rice embryos. These genes are known to be feedback regulated by the hydrolytic products of starchy endosperm such as the simple sugar glucose. It was found that oxygen deficiency interferes with the repression of Amy3D gene expression imposed by low concentrations of glucose but not with that imposed by higher amounts. This differential anoxic de-repression depending on sugar concentration suggests the presence of two distinct pathways for sugar regulation of Amy3D gene expression. Anoxic de-repression can be mimicked by treating rice embryos with inhibitors of ATP synthesis during respiration. Other sugar-regulated rice alpha-amylase genes, Amy3B/C and 3E, behave similarly to Amy3D. Treatment with a respiratory inhibitor or anoxia also relieved the sugar repression of the rice CIPK15 gene, a main upstream positive regulator of SnRK1A that is critical for Amy3D expression in response to sugar starvation. SnRK1A accumulation was previously shown to be required for MYBS1 expression, which transactivates Amy3D by binding to a cis-acting element found in the proximal region of all Amy3 subfamily gene promoters (the TA box). Taken together, these results suggest that prevention of oxidative phosphorylation by oxygen deficiency interferes with the sugar repression of Amy3 subfamily gene expression, leading to their enhanced expression in rice embryos during anaerobic germination.