Interaction of a transcriptional repressor with the RNA polymerase II holoenzyme plays a crucial role in repression

The yeast transcriptional repressor Tup1, tethered to DNA, represses to strikingly different degrees transcription elicited by members of two classes of activators. Repression in both cases is virtually eliminated by mutation of either member of the cyclin-kinase pair Srb10/11. In contrast, telomeric chromatin affects both classes of activators equally, and in neither case is that repression affected by mutation of Srb10/11. In vitro, Tup1 interacts with RNA polymerase II holoenzyme bearing Srb10 as well as with the separated Srb10. These and other findings indicate that at least one aspect of Tup1's action involves interaction with the RNA polymerase II holoenzyme.

Induction and repression of DAN1 and the family of anaerobic mannoprotein genes in Saccharomyces cerevisiae occurs through a complex array of regulatory sites

The DAN/TIR mannoprotein genes of Saccharomyces cerevisiae (DAN1, DAN2, DAN3, DAN4, TIR1, TIR2, TIR3 and TIR4) are expressed in anaerobic cells while the predominant cell wall proteins Cwp1 and Cwp2 are down-regulated. Elements involved in activation and repression of the DAN/TIR genes were defined in this study, using the DAN1 promoter as a model. Nested deletions in a DAN1/lacZ reporter pinpointed regions carrying activation and repression elements. Inspection revealed two consensus sequences subsequently shown to be independent anaerobic response elements (AR1, consensus TCGTTYAG; AR2, consensus AAAAATTGTTGA). AR1 is found in all of the DAN/TIR promoters; AR2 is found in DAN1, DAN2 and DAN3. A 120 bp segment carrying two copies of AR1 preferentially activated transcription of lacZ under anaerobic conditions. A fusion of three synthetic copies of AR1 to MEL1 was also expressed anaerobically. Mutations in either AR1 site within the 120 bp segment caused a drastic loss of expression, indicating that both are necessary for activation and implying cooperativity between adjacent transcriptional activation complexes. A single AR2 site carried on a 46 bp fragment from the DAN1 promoter activated lacZ transcription under anaerobic conditions, as did a 26 bp synthetic AR2 fragment fused to MEL1. Nucleotide substitutions within the AR2 sequence eliminated the activity of the 46 bp segment. Ablation of the AR2 sequences in the full promoter caused a partial reduction of expression. The presence of the ATTGTT core (recognized by HMG proteins) in the AR2 sequence suggests that an HMG protein may activate through AR2. One region was implicated in aerobic repression of DAN1. It contains sites for the heme-induced Mot3 and Rox1 repressors.

Appetite of an epiphyte: Quantitative monitoring of bacterial sugar consumption in the phyllosphere

We report here the construction, characterization, and application of a bacterial bioreporter for fructose and sucrose that was designed to monitor the availability of these sugars to microbial colonizers of the phyllosphere. Plasmid pPfruB-gfp[AAV] carries the Escherichia coli fruB promoter upstream from the gfp[AAV] allele that codes for an unstable variant of green fluorescent protein (GFP). In Erwinia herbicola, this plasmid brings about the accumulation of GFP fluorescence in response to both fructose and sucrose. Cells of E. herbicola (pPfruB-gfp[AAV]) were sprayed onto bean plants, recovered from leaves at various time intervals after inoculation, and analyzed individually for GFP content by quantitative analysis of digital microscope images. We observed a positive correlation between single-cell GFP accumulation and ribosomal content as determined by fluorescence in situ hybridization, indicating that foliar growth of E. herbicola occurred at the expense of fructose and/or sucrose. One hour after inoculation, nearly all bioreporter cells appeared to be actively engaged in fructose consumption. This fraction dropped to approximately 11% after 7 h and to ≈1% a day after inoculation. This pattern suggests a highly heterogeneous availability of fructose to individual E. herbicola cells as they colonize the phyllosphere. We estimated that individual cells were exposed to local initial fructose abundances ranging from less than 0.15 pg fructose to more than 4.6 pg.

A Cross-Polarization, Magic-Angle-Spinning, 13C-Nuclear-Magnetic-Resonance Study of Polysaccharides in Sugar Beet Cell Walls1

Solid-state nuclear magnetic resonance relaxation experiments were used to study the rigidity and spatial proximity of polymers in sugar beet (Beta vulgaris) cell walls. Proton T1ρ decay and cross-polarization patterns were consistent with the presence of rigid, crystalline cellulose microfibrils with a diameter of approximately 3 nm, mobile pectic galacturonans, and highly mobile arabinans. A direct-polarization, magic-angle-spinning spectrum recorded under conditions adapted to mobile polymers showed only the arabinans, which had a conformation similar to that of beet arabinans in solution. These cell walls contained very small amounts of hemicellulosic polymers such as xyloglucan, xylan, and mannan, and no arabinan or galacturonan fraction closely associated with cellulose microfibrils, as would be expected of hemicelluloses. Cellulose microfibrils in the beet cell walls were stable in the absence of any polysaccharide coating.

Induction of male sterility in plants by metabolic engineering of the carbohydrate supply

Extracellular invertase mediates phloem unloading via an apoplastic pathway. The gene encoding isoenzyme Nin88 from tobacco was cloned and shown to be characterized by a specific spatial and temporal expression pattern. Tissue-specific antisense repression of Nin88 under control of the corresponding promoter in tobacco results in a block during early stages of pollen development, thus, causing male sterility. This result demonstrates a critical role of extracellular invertase in pollen development and strongly supports the essential function of extracellular sucrose cleavage for supplying carbohydrates to sink tissues via the apoplast. The specific interference with phloem unloading, the sugar status, and metabolic signaling during pollen formation will be a potentially valuable approach to induce male sterility in various crop species for hybrid seed production.

The H+-Sucrose Cotransporter NtSUT1 Is Essential for Sugar Export from Tobacco Leaves1

In many species translocation of sucrose from the mesophyll to the phloem is carrier mediated. A sucrose/H+-symporter cDNA, NtSUT1, was isolated from tobacco (Nicotiana tabacum) and shown to be highly expressed in mature leaves and at low levels in other tissues, including floral organs. To study the in vivo function of NtSUT1, tobacco plants were transformed with a SUT1 antisense construct under control of the cauliflower mosaic virus 35S promoter. Upon maturation, leaves of transformants expressing reduced amounts of SUT1 mRNA curled downward, and strongly affected plants developed chloroses and necroses that led to death. The leaves exhibited impaired ability to export recently fixed 14CO2 and were unable to export transient starch during extended periods of darkness. As a consequence, soluble carbohydrates accumulated and photosynthesis was reduced. Autoradiographs of leaves show a heterogenous pattern of CO2 fixation even after a 24-h chase. The 14C pattern does not change with time, suggesting that movement of photosynthate between mesophyll cells may also be impaired. The affected lines show a reduction in the development of the root system and delayed or impaired flowering. Taken together, the effects observed in a seed plant (tobacco) demonstrate the importance of SUT1 for sucrose loading into the phloem via an apoplastic route and possibly for intermesophyll transport as well.

Differential Regulation of Sugar-Sensitive Sucrose Synthases by Hypoxia and Anoxia Indicate Complementary Transcriptional and Posttranscriptional Responses1

The goal of this research was to resolve the hypoxic and anoxic responses of maize (Zea mays) sucrose (Suc) synthases known to differ in their sugar regulation. The two maize Suc synthase genes, Sus1 and Sh1, both respond to sugar and O2, and recent work suggests commonalities between these signaling systems. Maize seedlings (NK508 hybrid, W22 inbred, and an isogenic sh1-null mutant) were exposed to anoxic, hypoxic, and aerobic conditions (0, 3, and 21% O2, respectively), when primary roots had reached approximately 5 cm. One-centimeter tips were excised for analysis during the 48-h treatments. At the mRNA level, Sus1 was rapidly up-regulated by hypoxia (approximately 5-fold in 6 h), whereas anoxia had less effect. In contrast, Sh1 mRNA abundance increased strongly under anoxia (approximately 5-fold in 24 h) and was much less affected by hypoxia. At the enzyme level, total Suc synthase activity rose rapidly under hypoxia but showed little significant change during anoxia. The contributions of SUS1 and SH1 activities to these responses were dissected over time by comparing the sh1-null mutant with the isogenic wild type (Sus+, Sh1+). Sh1-dependent activity contributed most markedly to a rapid protein-level response consistently observed in the first 3 h, and, subsequently, to a long-term change mediated at the level of mRNA accumulation at 48 h. A complementary midterm rise in SUS1 activity varied in duration with genetic background. These data highlight the involvement of distinctly different genes and probable signal mechanisms under hypoxia and anoxia, and together with earlier work, show parallel induction of “feast and famine” Suc synthase genes by hypoxia and anoxia, respectively. In addition, complementary modes of transcriptional and posttranscriptional regulation are implicated by these data, and provide a mechanism for sequential contributions from the Sus1 and Sh1 genes during progressive onset of naturally occurring low-O2 events.

Osmotic Stress Induces Expression of Choline Monooxygenase in Sugar Beet and Amaranth1

Choline monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach (Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet (Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl2 = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mm salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family.

Regulation of Leaf Senescence by Cytokinin, Sugars, and Light : Effects on NADH-Dependent Hydroxypyruvate Reductase

The aim of this study was to investigate the interactions between cytokinin, sugar repression, and light in the senescence-related decline in photosynthetic enzymes of leaves. In transgenic tobacco (Nicotiana tabacum) plants that induce the production of cytokinin in senescing tissue, the age-dependent decline in NADH-dependent hydroxypyruvate reductase (HPR), ribulose-1,5-bisphosphate carboxylase/oxygenase, and other enzymes involved in photosynthetic metabolism was delayed but not prevented. Glucose (Glc) and fructose contents increased with leaf age in wild-type tobacco and, to a greater extent, in transgenic tobacco. To study whether sugar accumulation in senescing leaves can counteract the effect of cytokinin on senescence, discs of wild-type leaves were incubated with Glc and cytokinin solutions. The photorespiratory enzyme HPR declined rapidly in the presence of 20 mm Glc, especially at very low photon flux density. Although HPR protein was increased in the presence of cytokinin, cytokinin did not prevent the Glc-dependent decline. Illumination at moderate photon flux density resulted in the rapid synthesis of HPR and partially prevented the negative effect of Glc. Similar results were obtained for the photosynthetic enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase. It is concluded that sugars, cytokinin, and light interact during senescence by influencing the decline in proteins involved in photosynthetic metabolism.

Repression of TFII-I-dependent transcription by nuclear exclusion

TFII-I is an unusual transcription factor possessing both basal and signal-induced transcriptional functions. Here we report the characterization of a TFII-I-related factor (MusTRD1/BEN) that regulates transcriptional functions of TFII-I by controlling its nuclear residency. MusTRD1/BEN has five or six direct repeats, each containing helix–loop–helix motifs, and, thus, belongs to the TFII-I family of transcription factors. TFII-I and MusTRD1/BEN, when expressed individually, show predominant nuclear localization. However, when the two proteins are coexpressed ectopically, MusTRD1/BEN locates almost exclusively to the nucleus, whereas TFII-I is largely excluded from the nucleus, resulting in a loss of TFII-I-dependent transcriptional activation of the c-fos promoter. Mutation of a consensus nuclear localization signal in MusTRD1/BEN results in a reversal of nuclear residency of the two proteins and a concomitant gain of c-fos promoter activity. These data suggest a means of transcriptional repression by competition at the level of nuclear occupancy.