Allele-Dependent Barley Grain β-Amylase Activity1

The wild ancestor of cultivated barley, Hordeum vulgare subsp. spontaneum (K. Koch) A. & Gr. (H. spontaneum), is a source of wide genetic diversity, including traits that are important for malting quality. A high β-amylase trait was previously identified in H. spontaneum strains from Israel, and transferred into the backcross progeny of a cross with the domesticated barley cv Adorra. We have used Southern-blot analysis and β-amy1 gene characterization to demonstrate that the high β-amylase trait in the backcross line is co-inherited with the β-amy1 gene from the H. spontaneum parent. We have analyzed the β-amy1 gene organization in various domesticated and wild-type barley strains and identified three distinct β-amy1 alleles. Two of these β-amy1 alleles were present in modern barley, one of which was specifically found in good malting barley cultivars. The third allele, linked with high grain β-amylase activity, was found only in a H. spontaneum strain from the Judean foothills in Israel. The sequences of three isolated β-amy1 alleles are compared. The involvement of specific intron III sequences, in particular a 126-bp palindromic insertion, in the allele-dependent expression of β-amylase activity in barley grain is proposed.

A Common Position-Dependent Mechanism Controls Cell-Type Patterning and GLABRA2 Regulation in the Root and Hypocotyl Epidermis of Arabidopsis1

A position-dependent pattern of epidermal cell types is produced during root development in Arabidopsis thaliana. This pattern is reflected in the expression pattern of GLABRA2 (GL2), a homeobox gene that regulates cell differentiation in the root epidermis. GL2 promoter::GUS fusions were used to show that the TTG gene, a regulator of root epidermis development, is necessary for maximal GL2 activity but is not required for the pattern of GL2 expression. Furthermore, GL2-promoter activity is influenced by expression of the myc-like maize R gene (35S::R) in Arabidopsis but is not affected by gl2 mutations. A position-dependent pattern of cell differentiation and GL2-promoter activity was also discovered in the hypocotyl epidermis that was analogous to the pattern in the root. Non-GL2-expressing cell files in the hypocotyl epidermis located outside anticlinal cortical cell walls exhibit reduced cell length and form stomata. Like the root, the hypocotyl GL2 activity was shown to be influenced by ttg and 35S::R but not by gl2. The parallel pattern of cell differentiation in the root and hypocotyl indicates that TTG and GL2 participate in a common position-dependent mechanism to control cell-type patterning throughout the apical-basal axis of the Arabidopsis seedling.

Calcium-Dependent Protein Phosphorylation May Mediate the Gibberellic Acid Response in Barley Aleurone1

Peptide substrates of well-defined protein kinases were microinjected into aleurone protoplasts of barley (Hordeum vulgare L. cv Himalaya) to inhibit, and therefore identify, protein kinase-regulated events in the transduction of the gibberellin (GA) and abscisic acid signals. Syntide-2, a substrate designed for Ca2+- and calmodulin (CaM)-dependent kinases, selectively inhibited the GA response, leaving constitutive and abscisic acid-regulated events unaffected. Microinjection of syntide did not affect the GA-induced increase in cytosolic [Ca2+], suggesting that it inhibited GA action downstream of the Ca2+ signal. When photoaffinity-labeled syntide-2 was electroporated into protoplasts and cross-linked to interacting proteins in situ, it selectively labeled proteins of approximately 30 and 55 kD. A 54-kD, soluble syntide-2 phosphorylating protein kinase was detected in aleurone cells. This kinase was activated by Ca2+ and was CaM independent, but was inhibited by the CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (250 μm), suggesting that it was a CaM-domain protein kinase-like activity. These results suggest that syntide-2 inhibits the GA response of the aleurone via an interaction with this kinase, implicating the 54-kD kinase as a Ca2+-dependent regulator of the GA response in these cells.

Guard Cells Possess a Calcium-Dependent Protein Kinase That Phosphorylates the KAT1 Potassium Channel1

Increasing evidence suggests that changes in cytosolic Ca2+ levels and phosphorylation play important roles in the regulation of stomatal aperture and as ion transporters of guard cells. However, protein kinases responsible for Ca2+ signaling in guard cells remain to be identified. Using biochemical approaches, we have identified a Ca2+-dependent protein kinase with a calmodulin-like domain (CDPK) in guard cell protoplasts of Vicia faba. Both autophosphorylation and catalytic activity of CDPK are Ca2+ dependent. CDPK exhibits a Ca2+-induced electrophoretic mobility shift and its Ca2+-dependent catalytic activity can be inhibited by the calmodulin antagonists trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide. Antibodies to soybean CDPKα cross-react with CDPK. Micromolar Ca2+ concentrations stimulate phosphorylation of several proteins from guard cells; cyclosporin A, a specific inhibitor of the Ca2+-dependent protein phosphatase calcineurin enhances the Ca2+-dependent phosphorylation of several soluble proteins. CDPK from guard cells phosphorylates the K+ channel KAT1 protein in a Ca2+-dependent manner. These results suggest that CDPK may be an important component of Ca2+ signaling in guard cells.

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.

Redox-dependent activation of CO dehydrogenase from Rhodospirillum rubrum

Studies of initial activities of carbon monoxide dehydrogenase (CODH) from Rhodospirillum rubrum show that CODH is mostly inactive at redox potentials higher than −300 mV. Initial activities measured at a wide range of redox potentials (0–500 mV) fit a function corresponding to the Nernst equation with a midpoint potential of −316 mV. Previously, extensive EPR studies of CODH have suggested that CODH has three distinct redox states: (i) a spin-coupled state at −60 to −300 mV that gives rise to an EPR signal termed Cred1; (ii) uncoupled states at <−320 mV in the absence of CO2 referred to as Cunc; and (iii) another spin-coupled state at <−320 mV in the presence of CO2 that gives rise to an EPR signal termed Cred2B. Because there is no initial CODH activity at potentials that give rise to Cred1, the state (Cred1) is not involved in the catalytic mechanism of this enzyme. At potentials more positive than −380 mV, CODH recovers its full activity over time when incubated with CO. This reductant-dependent conversion of CODH from an inactive to an active form is referred to hereafter as “autocatalysis.” Analyses of the autocatalytic activation process of CODH suggest that the autocatalysis is initiated by a small fraction of activated CODH; the small fraction of active CODH catalyzes CO oxidation and consequently lowers the redox potential of the assay system. This process is accelerated with time because of accumulation of the active enzyme.

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.

Related homing endonucleases I-BmoI and I-TevI use different strategies to cleave homologous recognition sites

A typical homing endonuclease initiates mobility of its group I intron by recognizing DNA both upstream and downstream of the intron insertion site of intronless alleles, preventing the endonuclease from binding and cleaving its own intron-containing allele. Here, we describe a GIY-YIG family homing endonuclease, I-BmoI, that possesses an unusual recognition sequence, encompassing 1 base pair upstream but 38 base pairs downstream of the intron insertion site. I-BmoI binds intron-containing and intronless substrates with equal affinity but can nevertheless discriminate between the two for cleavage. I-BmoI is encoded by a group I intron that interrupts the thymidylate synthase (TS) gene (thyA) of Bacillus mojavensis s87-18. This intron resembles one inserted 21 nucleotides further downstream in a homologous TS gene (td) of Escherichia coli phage T4. I-TevI, the T4 td intron-encoded GIY-YIG endonuclease, is very similar to I-BmoI, but each endonuclease gene is inserted within a different position of its respective intron. Remarkably, I-TevI and I-BmoI bind a homologous stretch of TS-encoding DNA and cleave their intronless substrates in very similar positions. Our results suggest that each endonuclease has independently evolved the ability to distinguish intron-containing from intronless alleles while maintaining the same conserved recognition sequence centered on DNA-encoding active site residues of TS.

Light-induced phase-delay of the chicken pineal circadian clock is associated with the induction of cE4bp4, a potential transcriptional repressor of cPer2 gene

The chicken pineal gland contains the autonomous circadian oscillator together with the photic-input pathway. We searched for chicken pineal genes that are induced by light in a time-of-day-dependent manner, and isolated chicken homolog of bZIP transcription factor E4bp4 (cE4bp4) showing high similarity to vrille, one of the Drosophila clock genes. cE4bp4 was expressed rhythmically in the pineal gland with a peak at very early (subjective) night under both 12-h light/12-h dark cycle and constant dark conditions, and the phase was nearly opposite to the expression rhythm of cPer2, a chicken pineal clock gene. Luciferase reporter gene assays showed that cE4BP4 represses cPer2 promoter through a E4BP4-recognition sequence present in the 5′-flanking region, indicating that cE4BP4 can down-regulate the chick pineal cPer2 expression. In vivo light-perturbation studies showed that the prolongation of the light period to early subjective night maintained the high level expression of the pineal cE4bp4, and presumably as a consequence delayed the onset of the induction of the pineal cPer2 expression in the next morning. These light-dependent changes in the mRNA levels of the pineal cE4bp4 and cPer2 were followed by a phase-delay of the subsequent cycles of cE4bp4/cPer2 expression, suggesting that cE4BP4 plays an important role in the phase-delaying process as a light-dependent suppressor of cPer2 gene.

An AU-box motif upstream of the SD sequence of light-dependent psbA transcripts confers mRNA instability in darkness in cyanobacteria

The psbA2 gene of a unicellular cyanobacterium, Microcystis aeruginosa K-81, encodes a D1 protein homolog in the reaction center of photosynthetic Photosystem II. The expression of the psbA2 transcript has been shown to be light-dependent as assessed under light and dark (12/12 h) cycling conditions. We aligned the 5′-untranslated leader regions (UTRs) of psbAs from different photosynthetic organisms and identified a conserved sequence, UAAAUAAA or the ‘AU-box’, just upstream of the SD sequences. To clarify the role of 5′-upstream cis-elements containing the AU-box for light-dependent expression of psbA2, a series of deletion and point mutations in the region were introduced into the genome of heterologous cyanobacterium Synechococcus sp. strain PCC 7942, and psbA2 expression was examined. A clear pattern of light-dependent expression was observed in recombinant cyanobacteria carrying the K-81 psbA2 –38/+36 region (which includes the minimal promoter element and a light-dependent cis-element with the AU-box), +1 indicating the transcription start site. A constitutive pattern of expression, in which the transcripts remained almost stable under dark conditions, was obtained in cells harboring the –38/+14 region (the minimal element), indicating that the +14/+36 region with the AU-box is important for the observed light-dependent expression. Point mutations analyses within the AU-box also revealed that changes in number, direction and identity (as assayed by adenine/uridine nucleotide substitutions) influenced the light-dependent pattern of expression. The level of psbA2 transcripts increased markedly in CG- or deletion-box mutants in the dark, strongly indicating that the AU- (AT-) box acts as a negative cis-element. Furthermore, characterization of transcript accumulation in cells treated with rifampicin suggests that psbA2 5′-mRNA is unstable in the dark, supporting the view that the light-dependent expression is controlled at the post-transcriptional level. We discuss various mechanisms that may lead to altered mRNA stability such as the binding of factor(s) or ribosomes to the 5′-UTR and possible roles of the AU-box motif and the SD sequence.

Perturbation of the Nucleus: A Novel Hog1p-independent, Pkc1p-dependent Consequence of Hypertonic Shock in Yeast

Hypertonic shock of Saccharomyces cerevisiae activates the Hog1p MAP kinase cascade. In contrast, protein kinase C (Pkc1p) and the “cell integrity” MAP kinase cascade are critical for the response to hypotonic shock. We observed that hypertonic shock transiently relocated many, but not all, nuclear and nucleolar proteins to the cytoplasm. We hypothesized that the relocation of nuclear proteins was due to activation of the Hog1p kinase cascade, yet, surprisingly, Hog1p was not required for these effects. In contrast, Pkc1p kinase activity was required, although the Pkc1p MAP kinase cascade and several factors known to lie upstream and downstream of Pkc1p were not. Moreover, sudden induction of a hyperactive form of Pkc1p was sufficient to relocate nuclear proteins. Taken together, these observations show that the scope of involvement of Pkc1p in the organization of the nucleus considerably exceeds what has been characterized previously. The relocation of nuclear proteins is likely to account for the profound inhibition of RNA synthesis that was observed during hypertonic shock.

A yeast gene, MGS1, encoding a DNA-dependent AAA+ ATPase is required to maintain genome stability

Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA+ class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability.

Mediator proteins orchestrate enzyme-ssDNA assembly during T4 recombination-dependent DNA replication and repair

Studies of recombination-dependent replication (RDR) in the T4 system have revealed the critical roles played by mediator proteins in the timely and productive loading of specific enzymes onto single-stranded DNA (ssDNA) during phage RDR processes. The T4 recombination mediator protein, uvsY, is necessary for the proper assembly of the T4 presynaptic filament (uvsX recombinase cooperatively bound to ssDNA), leading to the recombination-primed initiation of leading strand DNA synthesis. In the lagging strand synthesis component of RDR, replication mediator protein gp59 is required for the assembly of gp41, the DNA helicase component of the T4 primosome, onto lagging strand ssDNA. Together, uvsY and gp59 mediate the productive coupling of homologous recombination events to the initiation of T4 RDR. UvsY promotes presynaptic filament formation on 3′ ssDNA-tailed chromosomes, the physiological primers for T4 RDR, and recent results suggest that uvsY also may serve as a coupling factor between presynapsis and the nucleolytic resection of double-stranded DNA ends. Other results indicate that uvsY stabilizes uvsX bound to the invading strand, effectively preventing primosome assembly there. Instead, gp59 directs primosome assembly to the displaced strand of the D loop/replication fork. This partitioning mechanism enforced by the T4 recombination/replication mediator proteins guards against antirecombination activity of the helicase component and ensures that recombination intermediates formed by uvsX/uvsY will efficiently be converted into semiconservative DNA replication forks. Although the major mode of T4 RDR is semiconservative, we present biochemical evidence that a conservative “bubble migration” mode of RDR could play a role in lesion bypass by the T4 replication machinery.

Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer

Two major pathways of recombination-dependent DNA replication, “join-copy” and “join-cut-copy,” can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes 46 and 47) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.

ATP bound to the origin recognition complex is important for preRC formation

The origin recognition complex (ORC) binds origins of replication and directs the assembly of a higher order protein complex at these sites. ORC binds and hydrolyzes ATP in vitro. ATP binding to the largest subunit of ORC, Orc1p, stimulates specific binding to origin DNA; however, the function of ATP hydrolysis by ORC is unknown. To address the role of ATP hydrolysis, we have generated mutants within Orc1p that are dominant lethal. At physiological ATP concentrations, these mutants are defective for ATP hydrolysis but not ATP binding in the absence of DNA. These mutants inhibit formation of the prereplicative complex when overexpressed. The dominant lethal phenotype of these mutant ORC complexes is suppressed by simultaneous overexpression of wild-type, but not mutant, Cdc6p. Our findings suggest that these hydrolysis-defective mutants inhibit growth by titrating Cdc6p away from the origin. Based on these observations, we propose that Cdc6p specifically recognizes the ATP-bound state of Orc1p and that ATP hydrolysis is coupled to preRC disassembly.