bluuebunny/crossref_metadata_2025 huggingface dataset:
Stress induction and developmental regulation of a rice chitinase promoter in transgenic tobacco
Authors: Zhu Qun , Doerner Peter W. , Lamb Christopher J.
Published: March 2003

A 1.5 kb promoter fragment from the rice (Oryza sativa L.) RCH10 gene, which encodes a basic endochitinase inducible by wounding and fungal elicitor, was translationally fused to the β‐glucuronidase (GUS) reporter gene and transferred to tobacco by Agrobacterium tumefaciens‐mediated leaf disc transformation. Wounding of leaves induced GUS activity from low basal levels, and addition of fungal elicitor to the wounded tissue caused a further marked activation of the gene fusion. During vegetative development high levels of GUS activity were observed in roots and moderate levels in stems. Histochemical analysis indicated that the promoter was active in vascular and epidermal tissue, and the root apical tip. In flowers, high levels of GUS activity were observed in stigmas, ovaries and pollen‐containing anthers, but only low levels in sepals and petals. The promoter 5′‐deleted to −160 exhibited the same patterns of expression in floral organs, and was also strongly induced by wounding and elicitor, but GUS activity was markedly reduced in vegetative organs. More detailed 5′ deletions showed that a cis‐element required for floral expression was located between −160 and −74, and a cis element sufficient for stress induction was located 3′ of −74. This proximal region 3′ of −74 was also sufficient for expression in transfected rice protoplasts derived from suspension cultured cells. These data indicate that the complex developmental and environmental regulation of RCH10 promoter activity involves several distinct cis‐elements for vegetative expression, floral expression and stress induction, and that signal pathways for wound and elicitor induction are conserved between monocotyledonous and dicotyledonous plants.
The basic domain in the bZIP regulatory protein Opaque2 serves two independent functions: DNA binding and nuclear localization
Authors: Varagona Marguerite J. , Raikhel Natasha V.
Published: March 2003

For organisms to grow and develop, transcriptional regulatory proteins must localize to the nucleus. This movement is mediated by nuclear localization sequences (NLSs) which consist of short stretches of basic amino acids that are part of the structure of mature nuclear proteins. Two NLSs have been previously identified in the maize regulatory protein Opaque2 (O2), a member of the basic‐domain, leucine‐zipper (bZIP) class of proteins. In this report, it has been determined that both of these NLSs are necessary for import of O2, and the focus has been on a functional analysis of the more efficient, bipartite NLS that is present in the basic or DNA‐binding domain. A mutation which contains altered amino acids on both parts of this NLS severely reduced nuclear targeting, and led to the definition of two classes of bipartite NLSs. Since the bipartite NLS is located in the highly conserved, DNA‐binding domain, the dual role of this domain was examined using the maize mutant o2‐676, in which a conservative mutation eliminates the DNA‐binding function. The o2‐676 protein localized to the nucleus in maize and the bZIP domain from the mutant protein was sufficient to redirect a reporter protein to the nucleus in transgenic plant cells. Thus, it was possible to show that the nuclear targeting function of this domain is independent of DNA binding. Sequence conservation of the basic domain of other bZIP proteins suggests that the bifunctionality of this domain may be conserved in all members of this class; a consensus sequence for a bipartite NLS in bZIP proteins is suggested.
Flavonols and fertilization in Petunia hybrida: localization and mode of action during pollen tube growth
Authors: Ylstra Bauke , Busscher Jacqueline , Franken John , Hollman Peter C.H. , Mol Joseph N.M. , Van Tunen Arjen J.
Published: March 2003

Flavonols form an important class of flavonoids which serve an essential function during plant reproduction. Flavonoid biosynthesis is initiated by the enzyme chalcone synthase (CHS). A high abundance of flavonols and chs mRNA was demonstrated in male and female reproductive organs of Petunia hybrida. Detailed analyses revealed precise spatial and temporal regulation of the chs promoter and flavonol synthesis in the stigma, style and ovules. Transgenic plants were generated with a complete block of flavonol biosynthesis as the result of anti‐sense inhibition of chs gene activity. The absence of flavonols by this dominant mutation rendered these plants self‐sterile. Pollination experiments with wild‐type and mutant plants revealed that the production of flavonols in either the anthers or the pistils was required for pollen tube growth and seed set. Mutant pollen without flavonols in their exine germinated normally. However, after a short period of in vitro pollen tube growth the tips of these tubes disrupted and the protoplasm was disloaded leading to the death of the pollen grain. Addition of flavonol aglycones but not other flavonoids complemented this phenotype. Confocal laser scanning microscopy revealed the localization of high levels of flavonols throughout the wild‐type pollen tube. These compounds were not detected in the exine or cell wall of growing tubes. In addition, it was observed that the flavone apigenin could completely inhibit pollen tube growth. Taken together, it is shown that flavonols play an important role in the growth of the pollen tube and their mode of action is discussed.
Characterization of an Arabidopsis cDNA for a soluble epoxide hydrolase gene that is inducible by auxin and water stress
Authors: Kiyosue Tomohiro , Beetham Jeffrey K. , Pinot Frank , Hammock Bruce D. , Yamaguchi‐Shinozaki Kazuko , Shinozaki Kazuo
Published: March 2003

A cDNA (1122 bp) was isolated from a cDNA library prepared from Arabidopsis thaliana L. that had been subjected to drought stress for 1 h. The sequencing of a genomic clone corresponding to the cDNA and S1 mapping analysis revealed that the cDNA lacked the first 6 bp from its translational start (ATG). The resulting open reading frame encodes a polypeptide of 321 amino acids, and the calculated molecular weight of this polypeptide is 36 423 Da. The deduced amino acid sequence shows a high degree of similarity to C terminal halves of those of soluble epoxide hydrolases (sEHs) of human, mouse and rat, 35.5%, 34.1% and 33.1%, respectively. The cDNA was expressed in Escherichia coli cells, and the expressed protein migrates at 40 kDa when analyzed by SDS—PAGE. The recombinant protein at 40 kDa is much smaller than the mammalian sEH (58 kDa) but has characteristics of activity and inhibition similar to the mammalian sEHs when assayed with the substrate trans‐stilbene oxide and the inhibitors 4‐fluorochalcone oxide (4FCO), (2R,3R)‐3‐(4‐nitrophenyl) glycidol (RRNPG), and (2S,3S)‐3‐(4‐nitrophenyl)glycidol (SSNPG), which indicates that the cDNA did encode a soluble epoxide hydrolase of A. thaliana (AtsEH). Drought stress, but not heat or cold stress, slightly increased the accumulation of the mRNAs for AtsEH. The level of AtsEH transcripts increased strongly after treatment with a plant hormone, auxin (2,4‐dichlorophenoxyacetic acid, 2,4‐D; naphthaleneacetic acid, NAA; and indole‐3‐acetic acid, IAA) in young, pre‐bolting plants. Treatment with cytokinin (6‐benzylaminopurine, BA), abscisic acid (ABA) or gibberellin (GA3) had no detectable effect on AtsEH transcript levels. The transcripts for AtsEH gene were detected in the aerial vegetative organs of bolting plants (i.e. stems and leaves), but not in roots, flowers and seeds. The possible function of AtsEH is discussed. A similar sEH cDNA has recently been characterized in potato (Stapleton et al., 1994).
Auxin‐dependent microtubule responses and seedling development are affected in a rice mutant resistant to EPC
Authors: Nick Peter , Yatou Osamu , Furuya Masaki , Lambert Anne‐Marie
Published: March 2003

Mutants in rice (Oryza sativa L. cv. japonica) were used to study the role of the cytoskeleton in signal‐dependent morphogenesis. Mutants obtained by gamma ray irradiation were selected that failed to show inhibition of coleoptile elongation by the antimicrotubular drug ethyl‐N‐phenylcarbamate (EPC). The mutation EPC‐Resistant 31 (ER31), isolated from such a screen, caused lethality in putatively homozygous embryos. Heterozygotes exhibited drug resistance, impaired development of crown roots, and characteristic changes in the pattern of cell elongation: cell elongation was enhanced in mesocotyls and leaf sheaths, but inhibited in coleoptiles. The orientation of cortical microtubules changed correspondingly: for etiolated seedlings, compared with the wild‐type, they were more transverse with respect to the long cell axis in mesocotyls and leaf sheaths, but more longitudinal in coleoptiles. In mutant coleoptiles, in contrast to wild‐type, microtubules did not reorient in response to auxin, and their response to microtubule‐eliminating and microtubule‐stabilizing drugs was conspicuously reduced. In contrast, they responded normally to other stimuli such as gibberellins or red light. Auxin sensitivity as assayed by the dose‐response for callus induction did not show any significant differences between wild‐type and mutant. The mutant phenotype is interpreted in terms of an interrupted link between auxin‐triggered signal transduction and microtubule reorientation.
The Arabidopsis ribonuclease gene RNS1 is tightly controlled in response to phosphate limitation
Authors: Bariola Pauline A. , Howard Christie J. , Taylor Crispin B. , Verburg Michael T. , Jaglan Vanita D. , Green Pamela J.
Published: March 2003

Two stimuli that have been associated with nutrient remobilization in plants are phosphate (PI) starvation and senescence. Little is known about how the nutrient remobilization machinery is induced at the molecular level, but in the case of Pi starvation, ribonucleases are considered to play important roles in the remobilization process. Here, the control of two closely related ribonuclease genes of Arabidopsis, RNS1 and RNS3 is investigated. The RNS1 gene is sharply induced during starvation for Pi, an effect specific among the major macronutrients, whereas RNS3 transcript levels remain relatively constant. RNS1 and RNS3 produced in yeast co‐migrate with Arabidopsis ribonuclease activities that exhibit the same induction properties as the transcripts in both wild‐type plants and the pho1 mutant, which is defective in xylem loading of Pi. In contrast to what occurs during Pi starvation, both RNS1 and RNS3 are modestly induced during senescence, indicating that the two stimuli could trigger different signal transduction pathways. The characterization of RNS1, in particular, provides an important first step towards elucidating the mechanisms by which plants sense and respond to Pi limitation, a prominent condition in many soil types.
Real‐time imaging of phloem unloading in the root tip of Arabidopsis
Authors: Oparka K.J. , Duckett C.M. , Prior D.A.M. , Fisher D.B.
Published: March 2003

Confocal laser scanning microscopy (CLSM) has been used to image phloem transport and unloading in the root tip of Arabidopsis. The fluorescent probe 5(6) carboxyfluorescein (CF) was ester loaded into a single cotyledon and the entire seedling placed within an observation chamber under the microscope. Translocation of CF to the root tip was rapid, followed by unloading into discrete concentric files of cells. The position of the prominent unloading ‘zone’ corresponded precisely with that of the two protophloem files of sieve elements, demonstrating a functional role of these cells in symplastic sieve‐element unloading. Symplastic transport following unloading was confined to the elongating zone of the root with little basipetal transport to more mature cells. Following photobleaching of the unloading zone, phloem transport was restored immediately into the protophloem sieve elements, followed rapidly by lateral, symplastic sieve‐element unloading. The results demonstrate that phloem transport processes can now be imaged in real time, and non‐invasively, within an intact plant system.
Two of three promoter elements identified in a rice gene for proliferating cell nuclear antigen are essential for meristematic tissue‐specific expression
Authors: Kosugi Shunichi , Suzuka Iwao , Ohashi Yuko
Published: March 2003

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase δ, and it is highly conserved among eukaryotes. The rice PCNA promoter, truncated to position −263, was previously shown to confer meristematic tissue‐specific expression in transgenic plants. By DNasel footprinting and gel retardation analysis, three cis‐acting elements were defined in this truncated promoter, which designated site I (−201 to −194, CCAGGTGG), site IIa (−197 to −188, TGGGCCCGT) and site IIb (−178 to −169, TGGTCCCAC). Site I resembles the G‐box, and sites IIa and IIb resemble the conserved motif (T/GGTCCCAT) that is found in promoter regions of auxin‐regulated genes. Functional analysis of expression of a PCNA‐GUS gene fusion in transgenic tobacco plants revealed that a mutation in site I in the full‐length 2.0 kb promoter had no significant effect on the activity. However, the mutation in site I in the truncated −263 promoter, which had 14% of the activity of the full‐length promoter, caused a considerable decrease in the activity, suggesting that site I contributes in part to transcriptional activation. Simultaneous disruption of sites IIa and IIb in the full‐length promoter caused about 80–85% loss of promoter activity, while separate disruption of site IIa or site IIb resulted in no marked change on the activity. These observations suggest that site IIa and site IIb play an important role in the meristematic tissue‐specific expression of the rice PCNA gene, presumably by mediating putative enhancer activities dependent on the far‐upstream region.
Early cytological events after induction of cell division in egg cells and zygote development following in vitro fertilization with angiosperm gametes
Authors: Kranz Erhard , von Wiegen Petra , Lörz Horst
Published: March 2003

Early events, such as formation of the cell wall, first nuclear division and first unequal division of the zygote, were examined following in vitro fusion of single egg and sperm protoplasts of maize (Zea mays L.). The time course of these events was determined. The formation of cell wall components was observed 30 sec following egg—sperm fusion and proceeded continuously thereafter. Within 15 h after fusion most of the organelles became more densely grouped around the nucleus of the zygote. In the in vitro produced zygote the location of the cell organelles and of the dividing nucleus showed polarity. Two nucleoli were first observed 18 h after gamete fusion. The zygotic nucleus remained undivided for about 40 h. The first cell division was observed 40–60 h, generally 42–46 h, after egg—sperm fusion. The non‐fused egg cell could be triggered to sporophytic development in vitro by pulses of high amounts of 2,4‐D. Without such a treatment, cultured egg cells of different maize lines did not divide. Although nuclear fusion seemed to occur, fusion products of two egg cells also did not divide. Cell wall formation was incomplete and non‐uniform, showing a polarity of cultured egg cells and fusion products of two egg protoplasts. Cell division was also induced after fusion of maize egg with sperms of genetically remote species, such as Coix, Sorghum, Hordeum or Triticum. These gametic heterologous fusion products developed to microcalli. Moreover, cell division occurred in fusion products of an egg and a diploid somatic cell‐suspension protoplast from maize.
Developmental regulation of ribosomal protein L16 genes in Arabidopsis thaliana
Authors: Williams Mary E. , Sussex Ian M.
Published: March 2003

Lateral roots can be synchronously induced in Arabidopsis by a brief auxin treatment. An early event in the development of a lateral root primordium is the accumulation of mRNAs encoding ribosomal proteins. In situ hybridizations show that mRNA encoding one ribosomal protein, L16, accumulates in all rapidly proliferating tissues including the shoot and root apical meristems and lateral root primordia. To understand further the mechanisms by which ribosomal proteins are coordinately synthesized, two genes encoding the ribosomal protein L16 were isolated from Arabidopsis thaliana. Promoter sequences from each RPL16A and RPL16B were fused to the β‐glucuronidase reporter gene GUS. The promoter of RPL16B (from −848 to −19) conferred X‐Gluc staining in proliferating tissues including the shoot and root apical meristems. When GUS was expressed from the RPL16A promoter (from −875 to −22), X‐Gluc staining was observed in cells in the root stele and in anthers. When seedlings transformed with either promoter construct were treated with auxin to induce lateral roots, X‐Gluc staining accumulated in the lateral root primordia by 16 h after induction. Transcription of the RPL16B promoter appears to be correlated with cell division, while transcription of the RPL16A promoter is very cell specific. Expression of two genes encoding L16 during the early phase of lateral root initiation and in developing pollen may serve to increase levels of ribosomal proteins during the rapid growth of these tissues.
Heat‐inducible expression of FLP gene in maize cells
Authors: Lyznik Leszek A. , Hirayama Lynne , Rao K.V. , Abad Andre , Hodges Thomas K.
Published: March 2003

The soybean heat‐shock gene promoter (Gmhsp 17.5‐E) has been used to direct expression of gusA and FLP genes in maize cells. At inducible temperatures, in transient expression assays, gusA gene expression controlled by the heat‐shock promoter is about 10‐fold higher than the expression directed by the CaMV 35S promoter. The Gmhsp 17.5‐E promoter preserves its regulatory functions in heterologous maize cells after random integration into genomic DNA.

Heat‐shock inducible expression of the FLP gene was investigated by co‐transformation of the FLP expression vector (pHsFLP) and a recombination test vector (pUFNeo‐FmG) into maize protoplasts. Co‐transformed protoplasts were incubated at 42°C for 2 h. This treatment induced recombination of 20–25% of the available FRT sites in transient assays. As a result of heat‐shock treatment of stably co‐transformed maize cells, activation of gusA gene expression and an associated decrease or elimination of NPT‐II activity in transgenic maize lines was observed. Molecular evidence was obtained of the expected DNA excision process catalyzed by the FLP protein in maize transgenic cells. Thus, the experiments presented in this paper indicate that the FLP protein can recognize and subsequently recombine the FRT target sites that had integrated into plant genomic DNA, and that regulated expression of the FLP gene is possible in maize cells using the soybean heat‐shock promoter.
Isolation of two soybean G‐box binding factors which interact with a G‐box sequence of an auxin‐responsive gene
Authors: Hong Jong Chan , Cheong Yong Hwa , Nagao Ron T. , Bahk Jeong Dong , Key Joe L. , Cho Moo Je
Published: March 2003

G‐box binding factors (GBFs) constitute a family of plant DNA‐binding proteins that bind to the G‐box motif, a regulatory cis element present in many plant genes with a palindromic DNA motif of CACGTG. Previously TCCACGTGTC, a G‐box motif, from an auxin responsive gene GmAux28 has been identified as a sequence‐specific protein‐binding site. Here the isolation of two soybean cDNA clones, referred to as SGBF‐1 and SGBF‐2, encoding proteins which bind to the G‐box motif is reported. The primary structure of SGBF‐1 and SGBF‐2 predicts that these proteins contain a basic leucine zipper (bZIP) DNA‐binding domain and an N‐terminal proline‐rich domain.

A dramatic difference in the pattern of protein‐DNA complex formation was observed when recombinant SGBF‐1 and SGBF‐2 proteins were analyzed by electrophoretic mobility shift assays (EMSAs). The SGBF‐1 binding pattern obtained with the G‐box probe resulted in three major retarded bands while the SGBF‐2 formed a single complex. This shows that the characteristically diffuse banding pattern of plant nuclear proteins interacting with the G‐box is also observed in a binding assay using only one recombinant GBF. EMSAs were performed with a few selected binding sequences to study the effect of flanking nucleotides to the hexanucleotide G‐box core motif. The binding specificity of the SGBF proteins resembles that described for type A cauliflower nuclear G‐box binding proteins which bind class I G‐box elements [(G/T)(C/A)CACGTG(G/T)(A/C)]. Phylogenetic analysis of 13 GBF‐like proteins from various plant species reveals that the SGBF‐1 and SGBF‐2 proteins belong to different lineages, suggesting that they may have distinct functions in activating transcription.
Expression of an amino acid biosynthesis gene in tomato flowers: developmental upregulation and MeJa response are parenchyma‐specific and mutually compatible
Authors: Samach Alon , Broday Limor , Hareven Dana , Lifschitz Eliezer
Published: March 2003

The gene coding for threonine deaminase (TD), the enzyme which catalyzes the first committed step in the biosynthesis of isoleucine, was isolated from tomato as a consequence of its unusual 500‐fold upregulation in floral organs. It was subsequently shown that TD is induced in potato leaves in response to wounding, abscisic acid and methyl jasmonate (MeJa). Detailed analysis presented here, reveals an intricate developmental regulation pattern of gene expression in flowers that is operating solely in parenchyma territories. Yet, despite its high pre‐existing expression level, TD in flowers can be further induced by MeJa. Induction of TD in flowers as well as in leaves is effective only in the parenchyma domains, irrespective of the prior expression levels. TD is neither expressed nor induced in epidermal, vascular or sporogenous tissues. Promoter analysis in transgenic tomato plants indicates that induction of TD follows identical kinetics in flowers and leaves. Furthermore, the ‘conditioning’ of developmental upregulation in flowers, the response to MeJa in flowers and leaves, and the parenchyma‐specific expression are all mediated by the cis‐elements within the proximal 192 bp of the promoter. Promoter elements regulating the correct organ‐specific expression are located, however, further upstream. The promoter constructs used in this study can serve as useful tools for expressing extremely high levels of transgenes in specific cells. A scheme explaining tissue‐specific response to MeJa, in conjunction with developmental control, is discussed.
Use of RFLPs larger than 100 kbp to map the position and internal organization of the nucleolus organizer region on chromosome 2 in Arabidopsis thaliana
Authors: Copenhaver Gregory P. , Doelling Jed H. , Gens J. Scott , Pikaard Craig S.
Published: March 2003

In Arabidopsis thaliana ribosomal RNA genes (rRNA genes or rDNA) are grouped in two nucleolus organizer regions (NORs) that together comprise approximately 6% of the genome. The map positions of the NORs relative to other genetic markers are unknown. It was found that the restriction endonuclease HindIII cuts once in some but not all rRNA genes to yield strain‐specific RFLPs of 100–700 kb that could be visualized by pulsed‐field gel electrophoresis and Southern blotting. The HindIII RFLPs of the A. thaliana strains Columbia and Landsberg segregated among recombinant inbred lines derived from a cross between these two strains. Linkage analysis placed the NOR bearing the polymorphic HindIII sites to the top of the upper arm of chromosome 2. The name NOR2 is suggested for this locus. HindIII‐bearing rRNA genes are interspersed with clusters of HindIII‐less genes throughout NOR2. The observed clustering is most consistent with unequal crossing‐over, or nearest‐neighbor gene conversion, as the mechanism(s) that spread rRNA gene variants throughout an NOR. No meiotic cross‐over events yielding a ‘hybrid’ NOR with multiple RFLPs from both parents were observed among the 47 recombinant inbred lines examined. However, the appearance of novel HindIII profiles in approximately 40% of the recombinant inbred lines demonstrates that fluctuations in the distribution of rRNA gene variants occur frequently and can be readily detected on pulsed‐field gels.
Molecular characterization of a stigma‐specific gene encoding an arabinogalactan‐protein (AGP) from Nicotiana alata
Authors: Du He , Simpson Richard J. , Clarke Adrienne E. , Bacic Antony
Published: March 2003

Arabinogalactan‐proteins (AGPs) were isolated from the pistils of Nicotiana alata, deglycosylated, and the protein backbones fractionated by reversed‐phase HPLC as previously reported. A major fraction, RT35 was isolated and peptide sequences were obtained after protease digestion. A gene‐specific degenerate oligonucleotide was designed according to the amino acid sequences and a 380 bp PCR fragment was amplified in vitro from pistil RNA. The PCR fragment was used to screen a pistil cDNA library and a 762 bp cDNA clone (AGPNa3) was isolated and sequenced. The AGPNa3 cDNA encodes a 169 amino acid protein which consists of three domains: an N‐terminal secretion signal, a Pro‐rich domain and a C‐terminal Cys‐rich domain. The mature protein has 145 amino acid residues (16.7 kDa) and a predicted pl of 7.5. Northern blot analyses showed that the AGPNa3 gene was only expressed in the pistils of N. alata and of closely related Nicotiana species but not in other plants or suspension‐cultured cells. Further Northern blot analysis and in situ hybridization showed that within the pistil, it was primarily expressed in the stigmatic tissues of mature flowers.
Transcript processing internal to a mitochondrial open reading frame is correlated with fertility restoration in male‐sterile sorghum
Authors: Tang Hoang V. , Pring Daryl R. , Shaw Lynn C. , Salazar Reggie A. , Muza Figuhr R. , Yan Bin , Schertz Keith F.
Published: March 2003

A chimeric mitochondrial DNA (mtDNA) configuration of the cytoplasmic male‐sterile (cms) sorghum line IS1112C includes a 321 bp open reading frame designated orf107, encoding a predicted polypeptide product of 11.85 kDa. The open reading frame, similar to several other genes associated with cms, consists of amino‐terminal sequences derived from an obligate gene. Unlike other examples to date, however, the carboxy‐terminal sequences are highly similar to the carboxy terminus of an open reading frame implicated in cms of rice, orf79. The amino‐terminal 31 residues of orf107 are 84% similar to atp9, and the carboxy‐terminal 49 residues are 57% identical and 80% similar to the carboxy terminus of orf79. Transcripts of orf107 are edited, with four C‐to‐U changes that alter amino acids. Sorghum lines partially or fully restored to fertility exhibit a high‐efficiency internal‐orf107 transcript processing activity, precluding abundant whole‐length transcripts, while male‐sterile lines exhibit only a trace of the activity. Previous data on the abundance of a 12 kDa in organello‐synthesized polypeptide in male‐sterile versus male‐fertile lines are correlated with differential orf107 transcript processing activity of these lines. Examinations of backcross and F2 lines suggest a gametophytic mode of restoration, and indicate that enhanced transcript processing activity is necessary, but not sufficient, to restore full fertility. These novel observations indicate that mitochondrial open reading frames associated with cms in different species can include highly similar motifs, and that fertility restoration could involve a mechanism by which synthesis of a cms‐associated gene product may be precluded through internal transcript cleavage.
The Arabidopsis thaliana ACT4/ACT12 actin gene subclass is strongly expressed throughout pollen development
Authors: Huang Shurong , An Yong‐Qiang , McDowell John M. , McKinney Elizabeth C. , Meagher Richard B.
Published: March 2003

Plants contain complex actin gene families composed of several diverse and ancient subclasses of genes. One Arabidopsis actin gene subclass represented by the ACT4 and ACT12 genes has been isolated and characterized. Both actin genes have typical plant actin gene structures, including three small introns interrupting the coding region and an intron within the mRNA leader. Their encoded proteins differ from each other in only one amino acid, whereas they differ in 3–10% of their amino acids from the other five Arabidopsis actin subclasses. They also share a few small blocks of DNA sequence homology in the 5′ flanking region near their TATA boxs, but not in their introns, 3′ flanking regions, or degenerate positions within codons. Southern analysis with gene‐specific probes from 5′ flanking sequences showed that both were single copy genes in the genome. Both RNA gel blot analysis with 3′ gene‐specific probes and reverse transcriptase‐mediated polymerase chain reactions (RT‐PCR) with gene‐specific primers detected low levels of ACT4 and ACT12 mRNAs in flowers and very high levels in pollen. The RT‐PCR detected very low levels of these mRNAs in the vegetative organs. The 5′ region from both genes, including the promoter region, TATA box, the sequence for the mRNA leader and its intron, and the first 19 actin codons, was fused to a β‐glucuronidase (GUS) reporter gene. Expression of the GUS fusions were examined histochemically in 40 independent transgenic Arabidopsis plants. Expression of the ACT4/GUS fusion was restricted to young vascular tissues, tapetum, and developing and mature pollen. Similar expression patterns in these tissues and cell types were observed for ACT12/ GUS fusion, yet unlike ACT4, ACT12 was also strongly expressed in the root cap and in a ring of pericycle tissues during lateral root initiation and early development. The unique expression patterns of the ACT4/ACT12 actin gene subclass are discussed in light of recent data on the other expressed members of the Arabidopsis actin gene family.
Distinct isoforms of ADPglucose pyrophosphorylase occur inside and outside the amyloplasts in barley endosperm
Authors: Thorbjørnsen Tine , Villand Per , Denyer Kay , Olsen Odd‐Arne , Smith Alison M.
Published: March 2003

This paper addresses the controversial idea that ADPglucose pyrophosphorylase may be located in the cytosol in some non‐photosynthetic plant organs. The intracellular location of the enzyme in developing barley endosperm has been investigated by isolation of intact amyloplasts. Amyloplast preparations contained 13–17% of the total endosperm activity of two plastidial marker enzymes, and less than 0.5% of the total endosperm activity of two cytosolic marker enzymes. Amyloplast preparations contained about 2.5% of the ADPglucose pyrophosphorylase activity, indicating that approximately 15% of the ADPglucose pyrophosphorylase activity in young endosperms is plastidial. Immunoblotting of gels of endosperm and amyloplast extracts also indicated that the enzyme is both inside and outside the amyloplast. Antibodies to the small subunits of the enzyme from barley and maize revealed two bands of protein of different sizes, one of which was located inside and the other outside the amyloplast. The plastidial protein was of the same size as a protein in the chloroplasts of barley leaves which was also recognized by these antibodies. It is suggested that the barley plant contains two distinct isoforms of ADPglucose pyrophosphorylase: one located in plastids (chloroplasts and amyloplasts) and the other in the cytosol of the endosperm. The role of the cytosolic ADPglucose pyrophosphorylase is unknown. Although it may contribute ADPglucose to starch synthesis, the total activity of ADPglucose pyrophosphorylase in the endosperm is far in excess of the rate of starch synthesis and the plastidial isoform is probably capable of catalysing the entire flux of carbon to starch.
The Arabidopsis cyclin‐dependent kinase gene cdc2bAt is preferentially expressed during S and G2 phases of the cell cycle
Authors: Segers Gerda , Gadisseur Isabelle , Bergounioux Catherine , de Almeida Engler Janice , Jacqmard Annie , Van Montagu Marc , Inzé Dirk
Published: March 2003

Cell cycle progression is regulated by cyclin‐dependent kinases (CDKs). Arabidopsis thaliana contains two cdk genes, cdc2aAt and cdc2bAt. This paper compares the developmental and cell cycle phase‐dependent transcription of both cdk genes. In situ hybridizations revealed that cdc2bAt steady‐state mRNAs, much like cdc2aAt, are found both in meristematic cells and cells with a high proliferative competence. Cdc2aAt is expressed in every meristematic cell whereas cdc2bAt is found to be expressed in a patchy pattern. An even smaller number of meristematic cells express the mitotic cyc1At. These data indicate that cdc2bAt and cyc1At mRNAs accumulate in a particular cell cycle phase in agreement with evidence provided by hybridization experiments of flow cytometry‐sorted nuclei and the use of cell cycle blockers on roots. The data indicate that cdc2bAt is preferentially expressed in S and G2 phases whereas cdc2aAt expression is constitutive throughout the cell cycle, as shown previously. The existence of two distinct CDK classes in plants is proposed: (i) constitutively expressed CDKs containing a PSTAIRE motif (e.g. cdc2aAt) and (ii) CDKs with divergent motifs which are expressed during a limited interval of the cell cycle (e.g. cdc2bAt).
N‐linked oligosaccharide processing is not necessary for glycoprotein secretion in plants
Authors: Lerouge Patrice , Fitchette‐Lainé Anne‐Catherine , Chekkafi Aïcha , Avidgor Véronique , Faye Loïc
Published: March 2003

The role of N‐glycans in the secretion of glycoproteins by suspension‐cultured sycamore cells was studied. The transport of glycoproteins to the extracellular compartment was investigated in the presence of a glycan‐processing inhibitor, castanospermine. Castanospermine has been selected because it inhibits homogeneously glycan maturation in sycamore cells and leads to the accumulation of a single immature N‐glycan. The structure of this glycan has been identified as Glc3Man7GlcNAc2 by labeling experiments, affinity chromatography on concanavalin A‐Sepharose and proton NMR. In contrast with previous results showing that N‐glycosylation is a pre‐requisite for secretion of N‐linked glycoproteins, this secretion is not affected by the presence of castanospermine. As a consequence, the presence of this unprocessed glycan is sufficient for an efficient secretion of glycoproteins in the extracellular compartment of suspension‐cultured sycamore cells.
Controlling seed development and seed size in Vicia faba: a role for seed coat‐associated invertases and carbohydrate state
Authors: Weber Hans , Borisjuk Ljudmilla , Wobus Ulrich
Published: March 2003

We previously provided evidence that seed coat‐associated invertase is involved in controlling the carbohydrate state of developing seeds and, by this way, triggering developmental processes (Weber et al. (1995) Plant Cell, 7, 1835–1846). To verify our postulate, we compared seed development of two genotypes of Vicia faba differing in seed weight. The seed coat of the large‐seeded genotype formed a higher number of parenchymatous cell layers and matured later. VfCWINV1 encoding a cell wall‐bound invertase is expressed in the unloading zone of the seed coat. mRNA levels peaked later in ‘large’ coats and mRNA was present in more cell layers over a longer time period. Cell wall‐bound invertase activity revealed a similar accumulation pattern, obviously generating the high hexose conditions present in the endospermal cavity bathing the premature cotyledons and thus controlling their carbohydrate state. High hexose conditions were correlated with an extended mitotic activity of the ‘large’ cotyledons. In ‘large’ and ‘small’ cotyledons, sucrose levels rose when hexoses decreased apparently terminating cell divisions and initiating differentiation and storage activities. This developmental switch was delayed in ‘large’ embryos. To prove the outlined relationship, sucrose was added in vitro to mitotically active cotyledons. This treatment favoured nuclear expansion and starch accumulation over cell division. In contrast, a hexose‐based medium maintained cell divisions. We conclude that development of the embryo is coordinately regulated with that of the maternal seed coat which controls, by metabolic signals, the phase of cell division of the embryo and consequently also seed size.
Intron sequences are involved in the plastid‐ and light‐dependent expression of the spinach PsaD gene†
Authors: Bolle Cordelia , Herrmann Reinhold G. , Oelmüller Ralf
Published: March 2003

Plastid‐ and light‐regulated expression of the spinach PsaD gene in transgenic tobacco requires sequences downstream of the transcription start site, and promoter sequences alone are not sufficient to respond to these stimuli. The spinach PsaD mRNA level in transgenic tobacco is still plastid‐ and light‐responsive when the expression of the intron‐containing transcription unit is driven by the 35S RNA CaMV promoter indicating that PsaD contains (a) gene‐internal control element(s). If the genomic PsaD sequence in the latter construct was replaced by the cDNA, a constitutive expression of the PsaD transcript level was observed. It is concluded that the intron sequence contributes to the plastid‐ and light‐dependent expression of the spinach PsaD gene.
T‐DNA integration patterns in co‐transformed plant cells suggest that T‐DNA repeats originate from co‐integration of separate T‐DNAs
Authors: De Neve Myriam , De Buck Sylvie , Jacobs Anni , Van Montagu Marc , Depicker Ann
Published: March 2003

Nicotiana protoplasts and Arabidopsis leaf discs or roots were co‐cultivated with two Agrobacterium strains each carrying a different T‐DNA. Co‐transformed plants were selected and the integration of the different T‐DNAs was analysed at the genetic and genomic level. Genetic analysis showed that the T‐DNAs derived from different bacteria were frequently integrated at the same locus, independent of the plant species or transformation method used. Southern analysis revealed that 12 out of 27 Arabidopsis transformants contained the co‐transferred T‐DNAs linked to each other in all possible configurations but with a preference for those with at least one right border involved in linkage. Overall, our data support the hypothesis that ligation of separate T‐DNAs is a dominant mechanism in formation of the frequently observed repeats of identical T‐DNAs. We propose a scheme which could explain the formation of T‐DNA repeats and the preferential involvement of right borders in T‐DNA linkages.
Identification of an Arabidopsis locus required for resistance to turnip crinkle virus
Authors: Dempsey D’Maris Amick , Pathirana M. Sudam , Wobbe Kristin K. , Klessig Daniel F.
Published: March 2003

Inoculation of turnip crinkle virus (TCV) into a (TCV)‐resistant line of Arabidopsis thaliana, Di‐17, results in the development of a hypersensitive response (HR) on the inoculated leaves. In contrast, an HR does not occur when leaves of the TCV‐susceptible Di‐3 line or the susceptible ecotypes Columbia (Col‐0), or Landsberg erecta (Ler) are inoculated. Genetic analysis of progeny from crosses between Di‐17 and either Di‐3, Col‐0 or Ler demonstrates that the development of an HR is regulated by a single dominant nuclear locus, herein designated HRT. Using progeny from a Di‐17 X Col‐0 cross, HRT was mapped to chromosome 5, where it is tightly linked to the DFR locus. We also demonstrate that a variety of resistance‐associated phenomena, including the TCV‐induced accumulation of salicylic acid, camalexin and autofluorescent cell‐wall material, correlate with the HR, suggesting the possibility that HRT is required for their activation.
Transactivation of a target gene using a suppressor tRNA in transgenic tobacco plants
Authors: Choisne Nathalie , Martin‐Canadell Alfred , Small Ian
Published: March 2003

The abundance of tRNAs, together with their central role in translation, has generated considerable interest in the use of tRNA genes for biotechnological applications. One such application is the use of suppressor tRNAs to transactivate target genes containing premature stop codons. Previous work has shown that such systems can work in transient expression experiments in plant protoplasts; here these experiments are extended to show that suppression of stop codons can occur in whole plants. Transgenic tobacco plants homozygous for a modified tRNALeu gene expressing a strong amber suppressor tRNA, and plants carrying a β‐glucuronidase (gus) gene inactivated by a premature amber stop codon have been obtained. When the two types of plants are crossed, many of the F1 hybrids show significant GUS activity. The GUS activity is dependent on the presence of both the suppressor tRNA gene and the gus gene. Tobacco plants carrying the suppressor tRNA gene are phenotypically normal, fertile and the gene shows normal Mendelian inheritance. The potential applications of such a system are discussed.
Fast‐activating cation channel in barley mesophyll vacuoles. Inhibition by calcium
Authors: Tikhonova Lioubov I. , Pottosin Igor I. , Dietz Karl‐Josef , Schönknecht Gerald
Published: March 2003

In contrast to the vacuolar ion channels which are gated open by an increase of cytosolic Ca2+ the vacuolar ion currents at resting cytosolic Ca2+are poorly explored. Therefore, this study was performed to investigate the properties of the so‐called fast‐activating vacuolar (FV) current which dominates the electrical characteristics of the tonoplast at physiological free Ca2+ concentrations. Patch—clamp measurements were performed on whole barley (Hordeum vulgare) mesophyll vacuoles and on excised tonoplast patches. Single ion channels were identified, which, based on their selectivity, activation kinetics, Ca2+‐ and voltage‐dependence, carry the whole‐vacuole FV current. Reversal potential determinations indicated a K+ overs C− permeability ratio of about 30. Both inward and outward whole‐vacuole currents as well as the activity of single FV channels were inhibited by an increase of cytosolic Ca2+, with a Kd≈ 6 µM. At physiological vacuolar Ca2+ activities, the FV channel is an outward‐rectifying potassium channel. The FV channel was activated in less than a few milliseconds both by negative and positive potential steps, having a minimal activity that is 40 mV negative of the K+ equilibrium potential. It is proposed that transport of K+ through this cation channel controls the electrical potential difference across the tonoplast.
Structural cell‐wall proteins in protoxylem development: evidence for a repair process mediated by a glycine‐rich protein
Authors: Ryser Ulrich , Schorderet Martine , Zhao Gui‐Fang , Studer Daniel , Ruel Katia , Haul Gunter , Keller Beat
Published: March 2003

Polyclonal antibodies were used to localize structural cell‐wall proteins in differentiating protoxylem elements in etiolated bean and soybean hypocotyls at the light‐ and electron‐microscopic level. A proline‐rich protein was localized in the lignified secondary walls, but not in the primary walls of protoxylem elements, which remain unlignified, as shown with lignin‐specific antibodies. Secretion of the proline‐rich protein was observed during lignification in different cell types. A glycine‐rich protein (GRP1.8) was specifically localized in the modified primary walls of mature protoxylem elements and in cell corners between xylem elements and xylem parenchyma cells. The protein was secreted by Golgi bodies both in protoxylem cells after the lignification of their secondary walls and in the surrounding xylem parenchyma cells. The modified primary walls of protoxylem elements were visualized under the light microscope as filaments or sheets staining distinctly with the protein stain Coomassie blue. Electron micrographs of these walls show that they are composed of an amorphous material of moderate electron‐density and of polysaccharide microfibrils. These materials form a three‐dimensional network, interconnecting the ring‐ or spiral‐shaped secondary wall thickenings of protoxylem elements and xylem parenchyma cells. The results demonstrate that the modified primary walls of protoxylem cells are not simply breakdown products due to partial hydrolysis and passive elongation, as believed until now. Extensive repair processes produce cell walls with unique staining properties. It is concluded that these walls are unusually rich in protein and therefore have special chemical and physical properties.
High‐affinity binding site for ethylene‐inducing xylanase elicitor on Nicotiana tabacum membranes
Authors: Hanania Uri , Avni Adi
Published: March 2003

Challenge of Nicotiana tabacum cv Xanthi with the ethylene‐inducing xylanase (EIX) from Trichoderma viride causes rapid induction of plant defense responses leading to hypersensitive necrosis. This phenomenon is cultivar‐specific; no response is detected when N. tabacum cv Hicks is similarly treated. The responsiveness is determined in tobacco and tomato by a single dominant gene. EIX was labeled with fluorescein‐isothiocyanate and incubated with cell suspension cultures, protoplasts or microsomal membranes. Binding of EIX to the microsomal membranes was found to be specific and saturable, with a dissociation constant of 6.2 nM. Using confocal laser microscopy, the EIX binding site was localized to the plasma membrane. Binding of EIX to its high‐affinity site occurred in responsive species. These results demonstrate the existence of a high‐affinity binding site for EIX on the plasma membrane of responsive cultivars. Chemical cross‐linking of EIX to microsomal membranes from responding plants revealed a 66 kDa protein complex. This protein may function as the receptor that mediates the hypersensitive response induced by EIX binding.
Genetic characterization of late‐flowering traits induced by DNA hypomethylation mutation in Arabidopsis thaliana
Authors: Kakutani Tetsuji
Published: March 2003

Arabidopsis DNA hypomethylation mutation, ddm1, results in a variety of developmental abnormalities by slowly inducing heritable lesions at unlinked loci. Here, late‐flowering traits observed at high frequencies in independently‐established ddm1 lines were genetically characterized. In all of the four late‐flowering lines examined the traits were dominant and mapped to the same chromosomal region, which is close or possibly identical to the FWA locus. The ddm1‐induced phenotypic onsets are apparently not random mutation events, but specific to a group of genes, suggesting the underlying epigenetic mechanism. The DNA methylation mutant provide useful system for identifying epigenetically‐regulated genes important for plant development.
A pectate lyase from Zinnia elegans is auxin inducible
Authors: Domingo Concha , Roberts Keith , Stacey Nicola J. , Connerton Ian , Ruíz‐teran Francisco , McCann Maureen C.
Published: March 2003

The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to reproducibly trans‐differentiate into tracheary elements (TE) after 96 h, while in the presence of auxin alone the cells simply elongate. In a search for genes involved in modifications to cell‐wall architecture before any overt signs of cell differentiation, a differential hybridization of a 72‐h cDNA library with probes from mRNA at time‐points of 24 h and 72 h was done revealing a number of transcripts up‐regulated between these times. One of these cDNAs shows homology to pectate lyase, a pectin‐degrading enzyme. The complete cDNA sequence (ZePel) corresponds to a translated protein of 44 kDa with an N‐terminal signal peptide of about 2 kDa, and one potential N‐glycosylation site. Northern analysis confirms that the strong expression of this gene during TE induction occurs at a very early stage of the process and is due solely to the presence of auxin in the induction medium. In situ hybridization studies in young Zinnia stems show that ZePel expression is associated with vascular bundles and shoot primordia. Recombinant protein made in Escherichia coli possesses calcium‐dependent pectate lyase activity. Pectate lyase activity is detected in elongating and differentiating in vitro cell populations. The role of this enzyme in remodelling the cell wall during cell elongation and differentiation is discussed.