bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of cytokinins on the expression of phosphate starvation responsive genes in Arabidopsis
Authors: Martín Ana C. , Del Pozo Juan Carlos , Iglesias Joaquín , Rubio Vicente , Solano Roberto , De La Peña Alicia , Leyva Antonio , Paz‐Ares Javier
Published: March 2003

The increase in the ratio of root growth to shoot growth that occurs in response to phosphate (Pi) deprivation is paralleled by a decrease in cytokinin levels under the same conditions. However, the role of cytokinin in the rescue system for Pi starvation remains largely unknown. We have isolated a gene from Arabidopsis thaliana (AtIPS1) that is induced by Pi starvation, and studied the effect of cytokinin on its expression in response to Pi deprivation. AtIPS1 belongs to the TPSI1/Mt4 family, the members of which are specifically induced by Pi starvation, and the RNAs of which contain only short, non‐conserved open reading frames. Pi deprivation induces AtIPS1 expression in all cells of wild‐type plants, whereas in the pho1 mutant grown on Pi‐rich soils, AtIPS1 expression in the root was delimited by the endodermis. This supports the view that pho1 is impaired in xylem loading of Pi, and that long‐distance signals controlling the Pi starvation responses act via negative control. Exogenous cytokinins repress the expression of AtIPS1 and other Pi starvation‐responsive genes in response to Pi deprivation. However, cytokinins did not repress the increase in root‐hair number and length induced by Pi starvation, a response dependent on local Pi concentration rather than on whole‐plant Pi status. Our results raise the possibility that cytokinins may be involved in the negative modulation of long‐distance, systemically controlled Pi starvation responses, which are dependent on whole‐plant Pi status.
Expression of the plant cyclin‐dependent kinase inhibitor ICK1 affects cell division, plant growth and morphology
Authors: Wang Hong , Zhou Yongming , Gilmer Susan , Whitwill Steve , Fowke Larry C.
Published: March 2003

The plant CDK inhibitor ICK1 was identified previously from Arabidopis thaliana with its inhibitory activity characterized in vitro. ICK1 displayed several structural and functional features that are distinct from known animal CDK inhibitors. Despite the initial characterization, there is no information on the functions of any plant CDK inhibitor in plants. To gain insight into ICK1 functions in vivo and the role of cell division during plant growth and development, transgenic plants were generated expressing ICK1 driven by the cauliflower mosaic virus 35S promoter. In comparison to control plants, growth was significantly inhibited in transgenic 35S‐ICK1 plants, with some plants weighing <10% of wild‐type plants at the 3 week stage. Most organs of 35S‐ICK1 plants were smaller. There were also modifications in plant morphology such as shape and serration of leaves and petals. The changes were so drastic that 35S‐ICK1 plants with strong phenotype no longer resembled wild‐type plants morphologically. Analyses showed that increased ICK1 expression resulted in reduced CDK activity and reduced the number of cells in these plants. Cells in 35S‐ICK1 plants were larger than corresponding cells in control plants. These results demonstrate that ICK1 acts as a CDK inhibitor in the plant, and the inhibition of cell division by ICK1 expression has profound effects on plant growth and development. They also suggest that alterations of plant organ shape can be achieved by restriction of cell division.
A MADS domain gene involved in the transition to flowering in Arabidopsis
Authors: Borner Rony , Kampmann Grit , Chandler John , Gleißner Roland , Wisman Ellen , Apel Klaus , Melzer Siegbert
Published: March 2003

Flowering time in many plants is triggered by environmental factors that lead to uniform flowering in plant populations, ensuring higher reproductive success. So far, several genes have been identified that are involved in flowering time control. AGL20 (AGAMOUSLIKE20) is a MADS domain gene from Arabidopsis that is activated in shoot apical meristems during the transition to flowering. By transposon tagging we have identified late flowering agl20 mutants, showing that AGL20 is involved in flowering time control. In previously described late flowering mutants of the long‐day and constitutive pathways of floral induction the expression of AGL20 is down‐regulated, demonstrating that AGL20 acts downstream to the mutated genes. Moreover, we can show that AGL20 is also regulated by the gibberellin (GA) pathway, indicating that AGL20 integrates signals of different pathways of floral induction and might be a central component for the induction of flowering. In addition, the constitutive expression of AGL20 in Arabidopsis is sufficient for photoperiod independent flowering and the over‐expression of the orthologous gene from mustard, MADSA, in the classical short‐day tobacco Maryland Mammoth bypasses the strict photoperiodic control of flowering.
A novel cold‐inducible zinc finger protein from soybean, SCOF‐1, enhances cold tolerance in transgenic plants
Authors: Kim Jong Cheol , Lee Sang Hyoung , Cheong Yong Hwa , Yoo Cheol‐Min , Lee Soo In , Chun Hyun Jin , Yun Dae‐Jin , Hong Jong Chan , Lee Sang Yeol , Lim Chae Oh , Cho Moo Je
Published: March 2003

Cold stress on plants induces changes in the transcription of cold response genes. A cDNA clone encoding C2H2‐type zinc finger protein,SCOF‐1, was isolated from soybean. The transcription ofSCOF‐1is specifically induced by low temperature and abscisic acid (ABA) but not by dehydration or high salinity. Constitutive overexpression ofSCOF‐1induced cold‐regulated (COR) gene expression and enhanced cold tolerance of non‐acclimated transgenicArabidopsisand tobacco plants. SCOF‐1 localized to the nucleus but did not bind directly to either C‐repeat/dehydration (CRT/DRE) or ABA responsive element (ABRE),cis‐acting DNA regulatory elements present inCORgene promoters. However, SCOF‐1 greatly enhanced the DNA binding activity of SGBF‐1, a soybean G‐box binding bZIP transcription factor, to ABREin vitro. SCOF‐1 also interacted with SGBF‐1 in a yeast two‐hybrid system. The SGBF‐1 transactivated theβ‐glucuronidasereporter gene driven by the ABRE element inArabidopsisleaf protoplasts. Furthermore, the SCOF‐1 enhanced ABRE‐dependent gene expression mediated by SGBF‐1. These results suggest that SCOF‐1 may function as a positive regulator ofCORgene expression mediated by ABRE via protein–protein interaction, which in turn enhances cold tolerance of plants.
Analysis of PEAM4, the pea AP1 functional homologue, supports a model for AP1‐like genes controlling both floral meristem and floral organ identity in different plant species
Authors: Berbel Ana , Navarro Cristina , Ferrándiz Cristina , Cañas Luis Antonio , Madueño Francisco , Beltrán José‐Pío
Published: March 2003

APETALA1 (AP1) and its homologue SQUAMOSA (SQUA) are key regulatory genes specifying floral meristem identity in the model plants Arabidopsis and Antirrhinum. Despite many similarities in their sequence, expression and functions, only AP1 appears to have the additional role of specifying sepal and petal identity. No true AP1/SQUA‐functional homologues from any other plant species have been functionally studied in detail, therefore the question of how the different functions of AP1‐like genes are conserved between species has not been addressed. We have isolated and characterized PEAM4, the AP1/SQUA‐functional homologue from pea, a plant with a different floral morphology and inflorescence architecture to that of Arabidopsis or Antirrhinum. PEAM4 encodes for a polypeptide 76% identical to AP1, but lacks the C‐terminal prenylation motif, common to AP1 and SQUA, that has been suggested to control the activity of AP1. Nevertheless, constitutive expression of PEAM4 caused early flowering in tobacco and Arabidopsis. In Arabidopsis, PEAM4 also caused inflorescence‐to‐flower transformations similar to constitutive AP1 expression, and was able to rescue the floral organ defects of the strong ap1‐1 mutant. Our results suggest that the control of both floral meristem and floral organ identity by AP1 is not restricted to Arabidopsis, but is extended to species with diverse floral morphologies, such as pea.
Single guard cell recordings in intact plants: light‐induced hyperpolarization of the plasma membrane
Authors: Roelfsema M. Rob G. , Steinmeyer Ralf , Staal Marten , Hedrich Rainer
Published: March 2003

Guard cells are electrically isolated from other plant cells and therefore offer the unique possibility to conduct current‐ and voltage‐clamp recordings on single cells in an intact plant. Guard cells in their natural environment were impaled with double‐barreled electrodes and found to exhibit three physiological states. A minority of cells were classified as far‐depolarized cells. These cells exhibited positive membrane potentials and were dominated by the activity of voltage‐dependent anion channels. All other cells displayed both outward and inward rectifying K+‐channel activity. These cells were either depolarized or hyperpolarized, with average membrane potentials of −41 mV (SD 16) and −112 mV (SD 19), respectively. Depolarized guard cells extrude K+ through outward rectifying channels, while K+ is taken up via inward rectifying channels in hyperpolarized cells. Upon a light/dark transition, guard cells that were hyperpolarized in the light switched to the depolarized state. The depolarization was accompanied by a 35 pA decrease in pump current and an increase in the conductance of inward rectifying channels. Both an increase in pump current and a decrease in the conductance of the inward rectifier were triggered by blue light, while red light was ineffective. From these studies we conclude that light modulates plasma membrane transport through large membrane potential changes, reversing the K+‐efflux via outward rectifying channels to a K+‐influx via inward rectifying channels.
A novel gene mutation that confers abnormal patterns of β‐carotene accumulation in cauliflower (Brassica oleracea var. botrytis)
Authors: Li Li , Paolillo Dominick J. , Parthasarathy Mandayam V. , DiMuzio Elena M. , Garvin David F.
Published: March 2003

The Or gene of cauliflower (Brassica oleracea var. botrytis) causes many tissues of the plant to accumulate carotenoids and turn orange, which is suggestive of a perturbation of the normal regulation of carotenogenesis. A series of experiments to explore the cellular basis of the carotenoid accumulation induced by the Or gene was completed. The Or gene causes obvious carotenoid accumulation in weakly or unpigmented tissues such as the curd, pith, leaf bases and shoot meristems, and cryptically in some cells of other organs, including the roots and developing fruits. The dominant carotenoid accumulated is β‐carotene, which can reach levels that are several hundred‐fold higher than those in comparable wild‐type tissues. The β‐carotene accumulates in plastids mainly as a component of massive, highly ordered sheets. The Or gene does not affect carotenoid composition of leaves, nor does it alter color and chromoplast appearance in flower petals. Interestingly, mRNA from carotenogenic and other isoprenoid biosynthetic genes upstream of the carotenoid pathway was detected both in orange tissues of the mutant, and in comparable unpigmented wild‐type tissues. Thus the unpigmented wild‐type tissues are likely to be competent to synthesize carotenoids, but this process is suppressed by an unidentified mechanism. Our results suggest that the Or gene may induce carotenoid accumulation by initiating the synthesis of a carotenoid deposition sink in the form of the large carotenoid‐sequestering sheets.
Overexpression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis
Authors: Choe Sunghwa , Fujioka Shozo , Noguchi Takahiro , Takatsuto Suguru , Yoshida Shigeo , Feldmann Kenneth A.
Published: March 2003

Plants unable to synthesize or perceive brassinosteroids (BRs) are dwarfs. Arabidopsis dwf4 was shown to be defective in a steroid 22α hydroxylase (CYP90B1) step that is the putative rate‐limiting step in the BR biosynthetic pathway. To better understand the role of DWF4 in BR biosynthesis, transgenic Arabidopsis plants ectopically overexpressing DWF4 (AOD4) were generated, using the cauliflower mosaic virus 35S promoter, and their phenotypes were characterized. The hypocotyl length of both light‐ and dark‐grown AOD4 seedlings was increased dramatically as compared to wild type. At maturity, inflorescence height increased >35% in AOD4 lines and >14% in tobacco DWF4 overexpressing lines (TOD4), relative to controls. The total number of branches and siliques increased more than twofold in AOD4 plants, leading to a 59% increase in the number of seeds produced. Analysis of endogenous BR levels in dwf4, Ws‐2 and AOD4 revealed that dwf4 accumulated the precursors of the 22α‐hydroxylation steps, whereas overexpression of DWF4 resulted in increased levels of downstream compounds relative to Ws‐2, indicative of facilitated metabolic flow through the step. Both the levels of DWF4 transcripts and BR phenotypic effects were progressively increased in dwf4, wild‐type and AOD4 plants, respectively. This suggests that it will be possible to control plant growth by engineering DWF4 transcription in plants.
Involvement of a novel Arabidopsis phospholipase D, AtPLDδ, in dehydration‐inducible accumulation of phosphatidic acid in stress signalling
Authors: Katagiri Takeshi , Takahashi Seiji , Shinozaki Kazuo
Published: March 2003

Phospholipid metabolism is involved in plant responses to drought and salinity stress. To investigate the role of phospholipase D (PLD) and its product phosphatidic acid (PtdOH) in stress signalling, we isolated a novel PLD cDNA, designated AtPLDδ, by screening a cDNA library prepared from dehydrated Arabidopsis thaliana. The AtPLDδ protein, of 868 amino acids, has a putative catalytic domain and a C2 domain that is involved in Ca2+/phospholipid binding. The AtPLDδ mRNA accumulated in response to dehydration and high salt stress. Histochemical analysis showed that the AtPLDδ gene is strongly expressed in the vascular tissues of cotyledons and leaves under dehydration stress conditions. Under normal growth conditions, AtPLDδ was expressed in roots, leaves, stems and flowers but not in siliques. We showed that dehydration stimulates the accumulation of PtdOH. The accumulation of PtdOH in response to dehydration was significantly suppressed in AtPLDδ antisense transgenic plants. These results suggest that AtPLDδ may be involved in PtdOH accumulation in the dehydration stress response.
Localization of flavonoid enzymes in Arabidopsis roots
Authors: Saslowsky David , Winkel‐Shirley Brenda
Published: March 2003

Immunofluorescence and immuno‐electron microscopy have been used to test the hypothesis that flavonoid metabolism is organized as a membrane‐associated enzyme complex. The cellular and subcellular locations of chalcone synthase (CHS) and chalcone isomerase (CHI), the first two enzymes of this pathway, were examined in Arabidopsis roots. High levels of both enzymes were found in the epidermal and cortex cells of the elongation zone and the root tip, consistent with the accumulation of flavonoid endproducts at these sites. Co‐localization of CHS and CHI was observed at the endoplasmic reticulum and tonoplast in these cells, and also in electron‐dense regions that are, as yet, unidentified. In addition, a striking asymmetric distribution was observed for these enzymes in cortex cells of the elongation zone, which may provide clues about the physiological function of flavonoids in roots. The accumulation of CHS and CHI was also examined in tt7(88), a mutant in the gene for flavonoid 3′‐hydroxylase (F3′H), which has been postulated to serve as a membrane anchor for the flavonoid enzyme complex. CHS and CHI accumulated to lower levels in cortex cells and higher levels in epidermal cells in the roots of this mutant as compared with wild‐type plants. Moreover, the electron‐dense regions containing these two enzymes were not observed. However, localization of CHS and CHI to the ER and tonoplast did not appear to be affected, suggesting that other proteins may function in recruiting the ‘soluble’ flavonoid enzymes to membranes. Staining of flavonoid endproducts with DPBA was consistent with expression of CHS and CHI in these seedlings.
Development and commercial use of Bollgard®cotton in the USA – early promises versus today's reality
Authors: Perlak Frederick J. , Oppenhuizen Mark , Gustafson Karen , Voth Richard , Sivasupramaniam Saku , Heering David , Carey Boyd , Ihrig Robert A. , Roberts James K.
Published: March 2003

Bollgard®cotton is the trademark given to a number of varieties of cotton bio‐engineered to produce an insecticidal protein fromBacillus thuringiensis(Bt). When produced by the modified cotton plants, this protein controls certain lepidopterous cotton insect pests. Commercially available since 1996, these cotton varieties are purchased under a license agreement in which the growers pay a fee and agree to abide by the terms, which include a 1‐year license to use the technology and agreement to participate in an insect resistance management program. Today, Bollgard®cotton is grown on more than one‐third of all cotton acreage in the USA. This product has reduced cotton production costs and insecticide use by providing an effective alternative to chemical insecticides for the control of tobacco budworm,Heliothis virescens;cotton bollworm,Helicoverpa zea;and pink bollworm,Pectinophora gossypiella. The specificity and safety profile of the Bt protein producedin plantain cotton was maintained. It has retained its selectivity for lepidopterous insects and lacks the characteristics found in potential allergenic proteins. Fiber quality, the agronomic characteristics of the plant and seed composition remain unchanged. New cotton technology is being developed to provide improved insect control and a wider spectrum of activity. These future products could further reduce insecticide use in the production of cotton, while maintaining the high level of safety and reliability that has been demonstrated by five seasons of Bollgard®cotton use.
The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco
Authors: Aharoni Asaph , De Vos C. H. Ric , Wein Martina , Sun Zhongkui , Greco Raffaella , Kroon Arthur , Mol Joseph N. M. , O'Connell Ann P.
Published: March 2003

Fruit ripening is characterized by dramatic changes in gene expression, enzymatic activities and metabolism. Although the process of ripening has been studied extensively, we still lack valuable information on how the numerous metabolic pathways are regulated and co‐ordinated. In this paper we describe the characterization of FaMYB1, a ripening regulated strawberry gene member of the MYB family of transcription factors. Flowers of transgenic tobacco lines overexpressing FaMYB1 showed a severe reduction in pigmentation. A reduction in the level of cyanidin 3‐rutinoside (an anthocyanin) and of quercetin‐glycosides (flavonols) was observed. Expression of late flavonoid biosynthesis genes and their enzyme activities were aversely affected by FaMYB1 overexpression. Two‐hybrid assays in yeast showed that FaMYB1 could interact with other known anthocyanin regulators, but it does not act as a transcriptional activator. Interestingly, the C‐terminus of FaMYB1 contains the motif pdLNLD/ELxiG/S. This motif is contained in a region recently proposed to be involved in the repression of transcription by AtMYB4, an Arabidopsis MYB protein. Our results suggest that FaMYB1 may play a key role in regulating the biosynthesis of anthocyanins and flavonols in strawberry. It may act to repress transcription in order to balance the levels of anthocyanin pigments produced at the latter stages of strawberry fruit maturation, and/or to regulate metabolite levels in various branches of the flavonoid biosynthetic pathway.
A role for salicylic acid and NPR1 in regulating cell growth in Arabidopsis
Authors: Vanacker Hélène , Lu Hua , Rate Debra N. , Greenberg Jean T.
Published: March 2003

Salicylic acid (SA) plays a key role in activating defenses and cell death during plant–pathogen interactions. In response to some pathogens, SA also limits the extent of cell death, indicating that it acts positively or negatively depending on the host–pathogen interaction. In addition, we previously showed that SA affects cell growth in the Arabidopsis defense‐related mutants accelerated cell death 6–1 (acd6–1) and aberrant growth and death 2 (agd2). Using acd6–1, agd2 and two other defense‐related mutants, lesion simulating disease 6 (lsd6), suppressor of SA‐insensitivity (ssi1), we show here in detail that SA regulates cell growth by specifically affecting cell enlargement, endoreduplication and/or cell division. We find that SA can act either positively or negatively to regulate cell growth depending on the context in which signaling occurs. Additionally, Nonexpressor of PR 1 (NPR1), a key SA signaling protein important for regulating defenses and cell death, also acts to promote cell division and/or suppress endoreduplication during leaf development. We propose that SA interacts with multiple receptors or signaling pathways to control cellular alterations during normal development, pathogen attack and/or stress situations. We suggest that SA and NPR1 play broader roles in cell fate control than has previously been understood.
Graft transmission of induced and spontaneous post‐transcriptional silencing of chitinase genes
Authors: Crété Patrice , Leuenberger Sabrina , Iglesias Victor A. , Suarez Vivian , Schöb Hanspeter , Holtorf Hauke , Van Eeden Sjoerd , Meins Frederick
Published: March 2003

Sense and antisense tobacco chitinase (CHN) transgenes, Luciferase‐CHN transcriptional fusions, and promoterless CHN cDNAs were introduced biolistically into CHN transformants of tobacco that never exhibit spontaneous gene silencing. All of the constructs tested induced systemic silencing of the resident CHN transgene and endogenes. Nuclear run‐on transcription assays showed that local introduction of additional gene copies triggers systemic post‐transcriptional gene silencing (PTGS). Together, this provides evidence that additional transgene copies need not be either highly transcribed or produce sense transcripts to evoke production of systemic PTGS signals. CHN PTGS was transmitted by top grafting, but not by reciprocal grafting of mature stems or the exchange of tissue plugs. Thus, the commonly encountered difficulties in achieving graft‐transmission could reflect the method used. Silencing in sense but not antisense transformants was transmitted by grafting to a high‐expressing sense CHN scion suggesting that the elaboration of mobile signals may not be an essential feature of antisense‐mediated gene silencing.
Involvement of the Arabidopsisα‐DOX1 fatty acid dioxygenase in protection against oxidative stress and cell death
Authors: De León Inés Ponce , Sanz Ana , Hamberg Mats , Castresana Carmen
Published: March 2003

α‐dioxygenases (α‐DOXs) catalyze the primary oxygenation of fatty acids into a newly identified group of oxylipins. Here we show that expression of the Arabidopsis α‐DOX1 gene is induced in response to both incompatible and compatible bacterial infections. However, the level of α‐DOX1 mRNA and dioxygenase activity appears earlier and reaches higher values when infection promotes a hypersensitive reaction. Furthermore, whereas gene expression is confined to necrotic lesions during the hypersensitive response, it occurs throughout the chlorotic area during a compatible interaction. Accumulation of α‐DOX1 transcripts is impaired in SA‐compromised plants and induced by SA and by chemicals generating nitric oxide (NO), intracellular superoxide or singlet oxygen, three signals mediating host cell death. Transgenic plants with altered levels of α‐dioxygenase react like wild‐type plants to a compatible pathogen. In contrast, plants with reduced activity develop a more rapid and severe necrotic response than wild‐type plants to incompatible bacteria and paraquat treatment, respectively, and a milder response when α‐DOX1 is overproduced. Our results suggest that plant α‐dioxygenases are used to generate lipid‐derived molecules for a process that protects plant tissues from oxidative stress and cell death.
Arabidopsis glt1‐T mutant defines a role for NADH‐GOGAT in the non‐photorespiratory ammonium assimilatory pathway
Authors: Lancien Muriel , Martin Melinda , Hsieh Ming‐Hsiun , Leustek Tom , Goodman Howard , Coruzzi Gloria M.
Published: March 2003

The physiological role of the NADH‐dependent glutamine‐2‐oxoglutarate aminotransferase (NADH‐GOGAT) enzyme was addressed in Arabidopsis using gene expression analysis and by the characterization of a knock‐out T‐DNA insertion mutant (glt1‐T) in the single NADH‐GOGAT GLT1 gene. The NADH‐GOGAT GLT1 mRNA is expressed at higher levels in roots than in leaves. This expression pattern contrasts with GLU1, the major gene encoding Fd‐GOGAT, which is most highly expressed in leaves and is involved in photorespiration. These distinct organ‐specific expression patterns suggested a non‐redundant physiological role for the NADH‐GOGAT and Fd‐GOGAT gene products. To test the in vivo function of NADH‐GOGAT, we conducted molecular and physiological analysis of the glt1‐T mutant, which is null for NADH‐GOGAT, as judged by mRNA level and enzyme activity. Metabolic analysis showed that the glt1‐T mutant has a specific defect in growth and glutamate biosynthesis when photorespiration was repressed by 1% CO2. Under these conditions, the glt1‐T mutant displayed a 20% decrease in growth and a dramatic 70% reduction in glutamate levels. Herein, we discuss the significance of NADH‐GOGAT in non‐photorespiratory ammonium assimilation and in glutamate synthesis required for plant development.
Biochemical evidence for ubiquitin ligase activity of the Arabidopsis COP1 interacting protein 8 (CIP8)
Authors: Hardtke Christian S. , Okamoto Haruko , Stoop‐Myer Chatanika , Deng Xing Wang
Published: March 2003

Arabidopsis COP1 is a negative regulator of photomorphogenesis, which targets HY5, a positive regulator of photomorphogenesis, for degradation via the proteasome pathway in the absence of light. COP1 and its interactive partner CIP8 both possess RING finger motifs, characteristic of some E3 ubiquitin ligases. Here we show that CIP8 promotes ubiquitin attachment to HY5 in E2‐dependent fashion in vitro. CIP8 exhibits a strong interaction with the E2 enzyme AtUBC8 through its N‐terminal domain. Phosphorylation of HY5 by casein kinase II requires the beta subunit 2, but does not affect HY5′s susceptibility to ubiquitination. The RING domain of CIP8 is required but is not sufficient for ubiquitin ligase activity. Although the RING domain of CIP8 interacts with the RING domain of COP1, addition of recombinant COP1 fails to affect CIP8′s ubiquitin ligase activity towards HY5 in vitro. However, recombinant COP1 can pull‐down native CIP8 from the extract of dark‐grown seedlings, but not from the extract of light‐grown seedlings in a column‐binding assay, implying a requirement for light‐regulated modification in vivo. Our data suggest that CIP8 can form a minimal ubiquitin ligase in co‐operation with the E2 enzyme AtUBC8. It is possible that the AtUBC8‐CIP8 module might interact with COP1 in vivo, thereby participating in proteasome‐mediated degradation of HY5.
Antisense suppression of l‐galactose dehydrogenase in Arabidopsis thaliana provides evidence for its role in ascorbate synthesis and reveals light modulated l‐galactose synthesis
Authors: Gatzek Stephan , Wheeler Glen L. , Smirnoff Nicholas
Published: March 2003

l‐Galactose dehydrogenase (l‐GalDH), a novel enzyme that oxidizes l‐Gal to l‐galactono‐1,4‐lactone (l‐GalL), has been purified from pea seedlings and cloned from Arabidopsis thaliana. l‐GalL is a proposed substrate for ascorbate biosynthesis in plants, therefore the function of l‐GalDH in ascorbate biosynthesis was investigated by overexpression in tobacco and antisense suppression in A. thaliana. In tobacco the highest expressing lines had a 3.5‐fold increase in extractable activity, but this did not increase leaf ascorbate concentration. Arabidopsis thaliana, transformed with an antisense l‐GalDH construct, produced lines with 30% of wild‐type activity. These had lower leaf ascorbate concentration when grown under high light conditions. l‐Gal pool size increased in antisense transformants with low l‐GalDH activity, and l‐Gal concentration was negatively correlated with ascorbate. The results provide direct evidence for a role of l‐GalDH in ascorbate biosynthesis. Ascorbate pool size in A. thaliana is increased by acclimation to high light, but l‐GalDH expression was not affected. l‐Gal accumulation was higher in antisense plants acclimated to high light, indicating that the capacity to synthesize l‐Gal from GDP‐mannose is increased. Because the only known function of l‐GalL is ascorbate synthesis, these antisense plants provide an opportunity to investigate ascorbate function with minimal effects on carbohydrate metabolism. Measurements of other antioxidants revealed an increase in ascorbate‐ and pyrogallol‐dependent peroxidase activity in low‐ascorbate lines. As ascorbate is the major hydrogen peroxide‐scavenging antioxidant in plants, this could indicate a compensatory mechanism for controlling hydrogen peroxide concentration.
Conifer reproductive development involves B‐type MADS‐box genes with distinct and different activities in male organ primordia
Authors: Sundström Jens , Engström Peter
Published: March 2003

The Norway spruce MADS‐box genes DAL11, DAL12 and DAL13 are phylogenetically related to the angiosperm B‐function MADS‐box genes: genes that act together with A‐function genes in specifying petal identity and with C‐function genes in specifying stamen identity to floral organs. In this report we present evidence to suggest that the B‐gene function in the specification of identity of the pollen‐bearing organs has been conserved between conifers and angiosperms. Expression of DAL11 or DAL12 in transgenic Arabidopsis causes phenotypic changes which partly resemble those caused by ectopic expression of the endogenous B‐genes. In similar experiments, flowers of Arabidopsis plants expressing DAL13 showed a different homeotic change in that they formed ectopic anthers in whorls one, two or four. We also demonstrate the capacity of the spruce gene products to form homodimers, and that DAL11 and DAL13 may form heterodimers with each other and with the Arabidopsis B‐protein AP3, but not with PI, the second B‐gene product in Arabidopsis. In situ hybridization experiments show that the conifer B‐like genes are expressed specifically in developing pollen cones, but differ in both temporal and spatial distribution patterns. These results suggest that the B‐function in conifers is dual and is separated into a meristem identity and an organ identity function, the latter function possibly being independent of an interaction with the C‐function. Thus, even though an ancestral B‐function may have acted in combination with C to specify micro‐ and megasporangia, the B‐function has evolved differently in conifers and angiosperms.
Two distantly related genes encoding 1‐deoxy‐d‐xylulose 5‐phosphate synthases: differential regulation in shoots and apocarotenoid‐accumulating mycorrhizal roots
Authors: Walter Michael H. , Hans Joachim , Strack Dieter
Published: March 2003

Isopentenyl diphosphate, the universal precursor of isoprenoids, is synthesized by two separate routes, one in the cytosol and the other in plastids. The initial step of the plastidial pathway is catalysed by 1‐deoxy‐d‐xylulose 5‐phosphate synthase (DXS), which was previously thought to be encoded by a single‐copy gene. We have identified two distinct classes of DXS‐like cDNAs from the model legume Medicago truncatula. The deduced mature MtDXS1 and MtDXS2 proteins, excluding the predicted plastid‐targeting peptides, are similar in size (72.7 and 71.2 kDa) yet share only 70% identity in their amino acid sequences, and both encode functional DXS proteins as shown by heterologous expression in Escherichia coli. Available DXS sequences from other plants can easily be assigned to either class 1 or class 2. Partial sequences of multiple DXS genes in a single genome may be found in the databases of several monocot and dicot plants. Blot analyses of RNA from M. truncatula, maize, tomato and tobacco demonstrate preferential expression of DXS1 genes in many developing plant tissues except roots. By contrast, DXS2 transcript levels are low in most tissues but are strongly stimulated in roots upon colonization by mycorrhizal fungi, correlated with accumulation of carotenoids and apocarotenoids. Monoterpene‐synthesizing gland cells of leaf trichomes appear to be another site of DXS2 gene activity. The potential importance of DXS1 in many housekeeping functions and a still hypothetical role of DXS2 in the biosynthesis of secondary isoprenoids is discussed.
Insect‐resistant transgenic plants in a multi‐trophic context
Authors: Groot Astrid T. , Dicke Marcel
Published: March 2003

So far, genetic engineering of plants in the context of insect pest control has involved insertion of genes that code for toxins, and may be characterized as the incorporation of biopesticides into classical plant breeding. In the context of pesticide usage in pest control, natural enemies of herbivores have received increasing attention, because carnivorous arthropods are an important component of insect pest control. However, in plant breeding programmes, natural enemies of herbivores have largely been ignored, although there are many examples that show that plant breeding affects the effectiveness of biological control. Negative influences of modified plant characteristics on carnivorous arthropods may induce population growth of new, even more harmful pest species that had no pest status prior to the pesticide treatment. Sustainable pest management will only be possible when negative effects on non‐target, beneficial arthropods are minimized. In this review, we summarize the effects of insect‐resistant crops and insect‐resistant transgenic crops, especially Bt crops, from a food web perspective. As food web components, we distinguish target herbivores, non‐target herbivores, pollinators, parasitoids and predators. Below‐ground organisms such as Collembola, nematodes and earthworms should also be included in risk assessment studies, but have received little attention. The toxins produced in Bt plants retain their toxicity when bound to the soil, so accumulation of these toxins is likely to occur. Earthworms ingest the bound toxins but are not affected by them. However, earthworms may function as intermediaries through which the toxins are passed on to other trophic levels. In studies where effects of insect‐resistant (Bt) plants on natural enemies were considered, positive, negative and no effects have been found. So far, most studies have concentrated on natural enemies of target herbivores. However, Bt toxins are structurally rearranged when they bind to midgut receptors, so that they are likely to lose their toxicity inside target herbivores. What happens to the toxins in non‐target herbivores, and whether these herbivores may act as intermediaries through which the toxins may be passed on to the natural enemies, remains to be studied.
A novel cold‐inducible gene from Arabidopsis, RCI3, encodes a peroxidase that constitutes a component for stress tolerance
Authors: Llorente Francisco , López‐Cobollo Rosa María , Catalá Rafael , Martínez‐Zapater José Miguel , Salinas Julio
Published: March 2003

A cDNA from Arabidopsis corresponding to a new cold‐inducible gene, RCI3 (for Rare Cold Inducible gene 3), was isolated. Isoelectric focusing electrophoresis and staining of peroxidase activity demonstrated that RCI3 encodes an active cationic peroxidase. RNA‐blot analysis revealed that RCI3 expression in response to low temperature is negatively regulated by light, as RCI3 transcripts were exclusively detected in etiolated seedlings and roots of adult plants. RCI3 expression was also induced in etiolated seedlings, but not in roots, exposed to dehydration, salt stress or ABA, indicating that it is subjected to a complex regulation through different signaling pathways. Analysis of transgenic plants containing RCI3::GUS fusions established that this regulation occurs at the transcriptional level during plant development, and that cold‐induced RCI3 expression in roots is mainly restricted to the endodermis. Plants overexpressing RCI3 showed an increase in dehydration and salt tolerance, while antisense suppression of RCI3 expression gave dehydration‐ and salt‐sensitive phenotypes. These results indicate that RCI3 is involved in the tolerance to both stresses in Arabidopsis, and illustrate that manipulation of RCI3 has a potential with regard to plant improvement of stress tolerance.
Two long‐chain acyl‐CoA synthetases from Arabidopsis thaliana involved in peroxisomal fatty acid β‐oxidation
Authors: Fulda Martin , Shockey Jay , Werber Martin , Wolter Frank P. , Heinz Ernst
Published: March 2003

Post‐germinative growth of oilseeds is dependent on the breakdown of the stored lipid reserves. Long‐chain acyl‐CoA synthetase activities (LACS) are critically involved in this process by activating the released free fatty acids and thus feeding the β‐oxidation cycle in glyoxysomes. Here we report on the identification of two LACS genes, AtLACS6 and AtLACS7 from Arabidopsis thaliana coding for peroxisomal LACS proteins. The subcellular localization was verified by co‐expression studies of spectral variants of the green fluorescent protein (GFP). While AtLACS6 is targeted by a type 2 (PTS2) peroxisomal targeting sequence, for AtLACS7 a functional PTS1 as well as a PTS2 could be demonstrated. Possible explanations for this potentially redundant targeting information will be discussed. Expression studies of both genes revealed a strong induction 1 day after germination resembling the expression pattern of other genes involved in β‐oxidation. Analysis of the substrate specificities of the two LACS proteins demonstrated enzymatic activity for both enzymes with the whole spectrum of fatty acids found in stored lipid reserves. These results suggest that both LACS proteins might have overlapping functions and are able to initiate β‐oxidation in plant peroxisomes.
Nuclear genes that promote splicing of group I introns in the chloroplast 23S rRNA and psbA genes in Chlamydomonas reinhardtii
Authors: Li Fei , Holloway Stephen P. , Lee Jaesung , Herrin David L.
Published: March 2003

Single nucleotide substitutions were made in the core helices P4, P6, and P7, and in the metal‐binding GAAA motif in the J4/5 region of the chloroplast group I rRNA intron of Chlamydomonas reinhardtii, Cr.LSU. In vitro assays showed that these substitutions had surprisingly strong effects on Cr.LSU self‐splicing; however, splicing of all but the P6 mutations could be at least partially recovered by increasing the Mg2+ concentration. The mutant constructs were transformed into chloroplasts to replace the wild‐type intron; however, only the P4 mutants became homoplasmic, indicating that the other mutations were lethal. The splicing‐deficient P4125A mutant, which exhibited slow growth and light sensitivity, was used to isolate suppressor strains that showed a substantial restoration of Cr.LSU splicing. Genetic analysis of the 7151, 7120 and 71N1 suppressors indicated that these mutations are in at least two nuclear genes. The 7151 suppressor mutation, which defines the chloroplast‐splicing suppressor (css1) gene, had no obviously altered growth phenotype with the wild‐type intron, and was dominant in vegetative diploids containing the mutant intron. All three of the suppressor strains also suppressed a mutation in the P4 region of the fourth psbA intron, Cr.psbA4, indicating that these genes play a role in splicing of multiple group I introns in the chloroplast.
The auxin influx carrier is essential for correct leaf positioning
Authors: Stieger Pia A. , Reinhardt Didier , Kuhlemeier Cris
Published: March 2003

Auxin is of vital importance in virtually every aspect of plant growth and development, yet, even after almost a century of intense study, major gaps in our knowledge of its synthesis, distribution, perception, and signal transduction remain. One unique property of auxin is its polar transport, which in many well‐documented cases is a critical part of its mode of action. Auxin is actively transported through the action of both influx and efflux carriers. Inhibition of polar transport by the efflux inhibitorN‐1‐naphthylphthalamic acid (NPA) causes a complete cessation of leaf initiation, a defect that can be reversed by local application of the auxin, indole‐3‐acetic acid (IAA), to the responsive zone of the shoot apical meristem. In this study, we address the role of the auxin influx carrier in the positioning and outgrowth of leaf primordia at the shoot apical meristem of tomato. By using a combination of transport inhibitors and synthetic auxins, we demonstrate that interference with auxin influx has little effect on organ formation as such, but prevents proper localization of leaf primordia. These results suggest the existence of functional auxin concentration gradients in the shoot apical meristem that are actively set up and maintained by the action of efflux and influx carriers. We propose a model in which efflux carriers control auxin delivery to the shoot apical meristem, whereas influx and efflux carriers regulate auxin distribution within the meristem.
Mutation in domain II of IAA1 confers diverse auxin‐related phenotypes and represses auxin‐activated expression of Aux/IAA genes in steroid regulator‐inducible system
Authors: Park Jin‐Young , Kim Hye‐Joung , Kim Jungmook
Published: March 2003

Most of Aux/IAA genes are rapidly induced by auxin. The Aux/IAA proteins are short‐lived nuclear proteins sharing the four conserved domains. Domain II is critical for rapid degradation of Aux/IAA proteins. Among these gene family members, IAA1 is one of the earliest auxin‐inducible genes. We used a steroid hormone‐inducible system to reveal putative roles and downstream signaling of IAA1 in auxin response. Arabidopsis transgenic plants were generated expressing fusion protein of IAA1 (IAA1‐GR) or IAA1 with a mutation in domain II (iaa1‐GR) and the glucocorticoid hormone‐binding domain (GR). IAA1‐GR transgenic plants did not exhibit any discernable phenotypic differences by DEX treatment that allows nuclear translocation of the fusion protein. In contrast, diverse auxin‐related physiological processes including gravitropism and phototropism were impaired by DEX treatment in roots, hypocotyls, stems, and leaves in iaa1‐GR transgenic plants. Auxin induction of seven Aux/IAA mRNAs including IAA1 itself was repressed by DEX treatment, suggesting that IAA1 functions in the nucleus by mediating auxin response and might act as a negative feedback regulator for the expression of Aux/IAA genes including IAA1 itself. Auxin induction of Aux/IAA genes in the presence of cycloheximide can be repressed by DEX treatment, showing that the repression of transcription of the Aux/IAAs by the iaa1 mutant protein is primary. Wild‐type IAA1‐GR could not suppress auxin induction of IAA1 and IAA2. These results indicate that inhibition of auxin‐activated transcription of Aux/IAA genes by the iaa1 mutant protein might be responsible for alteration of various auxin responses.
pistillata‐5, anArabidopsisB class mutant with strong defects in petal but not in stamen development
Authors: Yang Yingzhen , Xiang Hongjun , Jack Thomas
Published: March 2003

TheArabidopsisfloral organ identity genesAPETALA3(AP3) andPISTILLATA(PI) encode related DNA‐binding proteins of the MADS family. Considerable evidence supports the hypothesis that a heterodimer of AP3 and PI is an essential component of B class activity. Allap3andpialleles characterized to date exhibit equivalent phenotypic defects in both whorls 2 and 3. In strongap3andpimutants, petals and stamens are missing and sepals and carpels develop in their place. Weakap3andpimutants exhibit partial conversions of petals to sepals and stamens to carpels. In this report, we describe the isolation and characterization ofpi‐5,an unusual B class mutant that exhibits defects in whorl 2 where sepals develop in place of petals, but third whorl stamens are most often normal.pi‐5flowers resemble those from 35S::SEP3 antisense plants.pi‐5contains missense mutation in the K domain (PIE125K). PIE125Kexhibits defects in heterodimerization with its partner protein AP3. Via a reverse yeast two‐hybrid screen, AP3K139Ewas isolated as a compensatory mutant of PIE125K. The compensatory interaction between PIE125Kand AP3K139Eis observed both in yeast two‐hybrid assays andin planta. On its own, AP3K139Eexhibits defects in specifying both petal and stamen identity. In addition, PIE125Kis defective in interaction with SEPALLATA proteins in both two‐ and three‐hybrid assays suggesting that PIE125Kis defective in forming higher order complexes of MADS proteins. The decreased concentration of PI/AP3/SEP complexes offers an explanation for the petal defects observed in bothpi‐5and 35S::SEP3 antisense plants.
Transcriptome analysis of sulfur depletion in Arabidopsis thaliana: interlacing of biosynthetic pathways provides response specificity
Authors: Nikiforova Victoria , Freitag Jens , Kempa Stefan , Adamik Monika , Hesse Holger , Hoefgen Rainer
Published: March 2003

Higher plants assimilate inorganic sulfate into cysteine, which is subsequently converted to methionine, and into a variety of other sulfur‐containing organic compounds. To resist sulfur deficiency, plants must demonstrate physiological flexibility: the expression of an extensive set of genes and gene regulators that act in the affected pathways or signalling cascades must be delicately tuned in response to environmental challenges. To elucidate this network of interactions, we have applied an array hybridisation/transcript profiling method to Arabidopsis plants subjected to 6, 10 and 13 days of constitutive and induced sulfur starvation. The temporal expression behaviour of approximately 7200 non‐redundant genes was analysed simultaneously. The experiment was designed in a way to identify statistically significant changes of gene expression based on sufficient numbers of repeated hybridisations performed with five uniform pools of plant material. The expression profiles were processed to select differentially expressed genes. Among the 1507 sulfur‐responsive clones implicated in this way, 632 genes responded specifically to sulfur deficiency by significant over‐expression. The sulfur‐responsive genes were grouped according to functional categories or biosynthetic pathways. As expected, genes of the sulfur assimilation pathway were altered in expression. Furthermore, genes involved in flavonoid, auxin, and jasmonate biosynthesis pathways were upregulated in conditions of sulfur deficiency. Based on the correlative analysis of gene expression patterns, we suggest that a complex co‐ordination of systematic responses to sulfur depletion is provided via integration of flavonoid, auxin and jasmonate pathway elements. Plait concept for transduction of specificity via the main non‐specific signalling stream is proposed.