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PubMed1996

Functional dissection of a bean chalcone synthase gene promoter in transgenic tobacco plants reveals sequence motifs essential for floral expression.

Expression of chalcone synthase (CHS), the first enzyme in the flavonoid branch of the phenylpropanoid biosynthetic pathway in plants, is induced by developmental cues and environmental stimuli. We used plant transformation technology to delineate the functional structure of the French bean CHS15 gene promoter during plant development. In the absence of an efficient transformation procedure for bean, Nicotiana tabacum was used as the model plant. CHS15 promoter activity, evaluated by measurements of beta-D-glucuronidase (GUS) activity, revealed a tissue-specific pattern of expression similar to that reported for CHS genes in bean. GUS activity was observed in flowers and root tips. Floral expression was confined to the pigmented part of petals and was induced in a transient fashion. Fine mapping of promoter cis-elements was accomplished using a set of promoter mutants generated by unidirectional deletions or by site-directed mutagenesis. Maximal floral and root-specific expression was found to require sequence elements located on both sides of the TATA-box. Two adjacent sequence motifs, the G-box (CACGTG) and H-box (CCTACC(N)7CT) located near the TATA-box, were both essential for floral expression, and were also found to be important for root-specific expression. The CHS15 promoter is regulated by a complex interplay between different cis-elements and their cognate factors. The conservation of both the G-box and H-box in different CHS promoters emphasizes their importance as regulatory motifs.

Acyltransferases↗
PubMed2006

Auxin transport and gravitational research: perspectives.

Gravity is a fundamental factor which affects all living organisms. Plant development is well adapted to gravity by directing roots downward and shoots upwards. For more than a century, plant biologists have been fascinated to describe the molecular mechanisms underlying the gravitropic response of plants. Important progress towards signal perception, transduction, and response has been made, but new tools are beginning to uncover the regulatory networks for gravitropic control. We summarise recent progress in study of gravitropism and discuss strategies to identify the molecular basis of the gravity response in Arabidopsis thaliana. This will put us on a road towards the molecular systems biology of the Arabidopsis gravitropic response.

Arabidopsis↗
PubMed2004

Tomato stress-responsive factor TSRF1 interacts with ethylene responsive element GCC box and regulates pathogen resistance to Ralstonia solanacearum.

Ethylene responsive factors (ERFs) are important in regulating plant pathogen resistance, abiotic stress tolerance and plant development. Recent studies have greatly enlarged the ERF protein family and revealed more important roles of ERFs in plants. Here, we report our finding of a tomato ERF protein TSRF1, which is transcriptionally up-regulated by ethylene, salicylic acid, or Ralstonia solanacearum strain BJ1057 infection. Biochemical analysis indicates that TSRF1 specifically interacts in vitro with the GCC box, an element present in the promoters of many pathogenesis-related (PR) genes. Further investigation evidences that TSRF1 activates in vivo the expression of reporter beta-glucuronidase gene controlled by GCC box. More importantly, overexpressing TSRF1 in tobacco and tomato constitutively activates the expression of PR genes, and subsequently enhancing transgenic plant resistance to the bacterial wilt caused by Ralstonia solanacearum strain BJ1057. Therefore our investigation not only extends the functions of ERF proteins in plant resistance to R. solanacearum, but also provides further clues to understanding the mechanism of host regulatory proteins in response to the infection of pathogens.

Amino Acid Sequence↗
PubMed2001

Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene.

A cDNA clone of a wheat germin-like oxalate oxidase (OxO) gene regulated by the constitutive CaMV 35S promoter was expressed in a hybrid poplar clone, Populus x euramericana ('Ogy'). Previous studies showed that OxO is likely to play an important role in several aspects of plant development, stress response, and defense against pathogens. In order to study this wheat oxalate oxidase gene in woody plants, the expression of this gene and the functions of the encoded enzyme were examined in vitro and in vivo in transgenic 'Ogy'. The enzyme activity in the transformed 'Ogy' was visualized by histochemical assays and in SDS-polyacrylamide gels. It was found that the wheat OxO gene is expressed in leaves, stems, and roots of the transgenic 'Ogy' plants and the encoded enzyme is able to break down oxalic acid. Transgenic 'Ogy' leaves were more tolerant to oxalic acid as well as more effective in increasing the pH in an oxalic acid solution when compared to untransformed controls. In addition, when leaf disks from 'Ogy' plants were inoculated with conidia of the poplar pathogenic fungus Septoria musiva, which produces oxalic acid, the OxO-transformed plants were more resistant than the untransformed controls.

Ascomycota↗
PubMed1996

A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis.

The KNOTTED class of plant genes encodes homeodomain proteins. These genes have been found in all plant species where they have been sought and, where examined, show expression patterns that suggest they play an important role in shoot meristem function. Until now, all mutant phenotypes associated with these genes have been due to gain-of-function mutations, making it difficult to deduce their wild-type function. Here we present evidence that the Arabidopsis SHOOT-MERISTEMLESS (STM) gene, required for shoot apical meristem formation during embryogenesis, encodes a class I KNOTTED-like protein. We also describe the expression pattern of this gene in the wild-type plant. To our knowledge, STM is the first gene shown to mark a specific pattern element in the developing plant embryo both phenotypically and molecularly.

Amino Acid Sequence↗
PubMed2005

Role of methylotrophy during symbiosis between Methylobacterium nodulans and Crotalaria podocarpa.

Some rare leguminous plants of the genus Crotalaria are specifically nodulated by the methylotrophic bacterium Methylobacterium nodulans. In this study, the expression and role of bacterial methylotrophy were investigated during symbiosis between M. nodulans, strain ORS 2060T, and its host legume, Crotalaria podocarpa. Using lacZ fusion to the mxaF gene, we showed that the methylotroph genes are expressed in the root nodules, suggesting methylotrophic activity during symbiosis. In addition, loss of the bacterial methylotrophic function significantly affected plant development. Indeed, inoculation of M. nodulans nonmethylotroph mutants in C. podocarpa decreased the total root nodule number per plant up to 60%, decreased the whole-plant nitrogen fixation capacity up to 42%, and reduced the total dry plant biomass up to 46% compared with the wild-type strain. In contrast, inoculation of the legume C. podocarpa with nonmethylotrophic mutants complemented with functional mxa genes restored the symbiotic wild phenotype. These results demonstrate the key role of methylotrophy during symbiosis between M. nodulans and C. podocarpa.

Crotalaria↗
PubMed1998

The H+-sucrose cotransporter NtSUT1 is essential for sugar export from tobacco leaves

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

Journal Article↗
PubMed2025

Cell-type-specific response to silicon treatment in soybean leaves revealed by single-nucleus RNA sequencing and targeted gene editing.

Mineral nutrient uptake and deposition profoundly influence plant development, stress resilience, and productivity. Silicon (Si), though classified as a non-essential element, significantly influences a plant's physiology, particularly in fortifying defense responses and mitigating stress. While the genetic and molecular mechanisms of Si uptake and transport are well studied in monocots, particularly rice, their role in dicot species, such as soybean, remains unclear at the cellular and molecular levels. In this study, we utilized single-nucleus RNA sequencing (snRNA-seq) to dissect cellular responses to Si accumulation in soybean leaves. We identified distinct cellular populations, including a unique Si-induced or Si-associated cell cluster within vascular cells, suggesting a specialized mechanism of Si distribution. Si treatment notably induced the expression of defense-related genes, with a pronounced enrichment in vascular cells, underscoring their pivotal role in activating plant defense mechanisms. Moreover, Si modulated the expression of genes involved in phytoalexin biosynthesis, salicylic acid, and immune receptor signaling, suggesting transcriptional priming of genes involved in defense responses. Further investigation of Si transporters revealed precise expression of an Si efflux gene in epidermal cells in response to Si treatment. We also validated the role of efflux Si transporters using a Xenopus oocyte assay and CRISPR/Cas9 genome editing of composite soybean plant roots. This study provides critical insights into the biotic stress regulatory networks influenced by Si treatment in soybean leaves at the single-cell level, thus laying the foundation for enhancing stress tolerance through optimized mineral nutrient uptake.

Glycine max↗
PubMed2003

DETERMINATE and LATE FLOWERING are two TERMINAL FLOWER1/CENTRORADIALIS homologs that control two distinct phases of flowering initiation and development in pea.

Genes in the TERMINAL FLOWER1 (TFL1)/CENTRORADIALIS family are important key regulatory genes involved in the control of flowering time and floral architecture in several different plant species. To understand the functions of TFL1 homologs in pea, we isolated three TFL1 homologs, which we have designated PsTFL1a, PsTFL1b, and PsTFL1c. By genetic mapping and sequencing of mutant alleles, we demonstrate that PsTFL1a corresponds to the DETERMINATE (DET) gene and PsTFL1c corresponds to the LATE FLOWERING (LF) gene. DET acts to maintain the indeterminacy of the apical meristem during flowering, and consistent with this role, DET expression is limited to the shoot apex after floral initiation. LF delays the induction of flowering by lengthening the vegetative phase, and allelic variation at the LF locus is an important component of natural variation for flowering time in pea. The most severe class of alleles flowers early and carries either a deletion of the entire PsTFL1c gene or an amino acid substitution. Other natural and induced alleles for LF, with an intermediate flowering time phenotype, present no changes in the PsTFL1c amino acid sequence but affect LF transcript level in the shoot apex: low LF transcript levels are correlated with early flowering, and high LF transcript levels are correlated with late flowering. Thus, different TFL1 homologs control two distinct aspects of plant development in pea, whereas a single gene, TFL1, performs both functions in Arabidopsis. These results show that different species have evolved different strategies to control key developmental transitions and also that the genetic basis for natural variation in flowering time may differ among plant species.

Amino Acid Sequence↗
PubMed2002

Expression analysis of a family of nsLTP genes tissue specifically expressed throughout the plant and during potato tuber life cycle.

Non-specific lipid-transfer proteins (nsLTPs) are capable of binding lipid compounds in plant tissues and are coded by the nsLTP genes. Here, we present the analysis of expression of a family of potato (Solanum tuberosum) nsLTP genes that express throughout the developing plant in a highly tissue-specific manner. Three transcript-derived fragments were isolated using an amplified restriction fragment polymorphism-derived technique for RNA fingerprinting that show homology to plant nsLTP genes. These transcript-derived fragments displayed modulated expression profiles related to the development of new tissues, with a peak of transcription around the time of tuberization and just prior to sprout development, at dormancy breakage. In addition, a homologous family of expressed sequence tags was identified whose individual members could be classified according to their tissue specificity. Two subgroups of expressed sequence tags were found to express during tuber life cycle. To study the regulation of potato nsLTP genes, two putative potato nsLTP promoters were isolated and their expression was studied using promoter-marker-gene fusions. The results showed that one of the two promoters directed a highly specific pattern of expression detected in the phloem surrounding the nodes of young plants and in the same tissue of tuber related organs, whereas the second putative promoter showed little tissue or organ specificity. This difference in expression is likely due to a 331-bp insertion present in the tissue-specific promoter.

Amino Acid Sequence↗
PubMed2001

Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth.

The distribution and biosynthesis of indole-3-acetic acid (IAA) was investigated during early plant development in Arabidopsis. The youngest leaves analysed, less than 0.5 mm in length, contained 250 pg mg(-1) of IAA and also exhibited the highest relative capacity to synthesize this hormone. A decrease of nearly one hundred-fold in IAA content occurred as the young leaves expanded to their full size, and this was accompanied by a clear shift in both pool size and IAA synthesis capacity. The correlation between high IAA content and intense cell division was further verified in tobacco leaves, where a detailed analysis revealed that dividing mesophyll tissue contained ten-fold higher IAA levels than tissue growing solely by elongation. We demonstrated that all parts of the young Arabidopsis plant can potentially contribute to the auxin needed for growth and development, as not only young leaves, but also all other parts of the plant such as cotyledons, expanding leaves and root tissues have the capacity to synthesize IAA de novo. We also observed that naphthylphthalamic acid (NPA) treatment induced tissue-dependent feedback inhibition of IAA biosynthesis in expanding leaves and cotyledons, but intriguingly not in young leaves or in the root system. This observation supports the hypothesis that there is a sophisticated tissue-specific regulatory mechanism for auxin biosynthesis. Finally, a strict requirement for maintaining the pool sizes of IAA was revealed as reductions in leaf expansion followed both decreases and increases in the IAA levels in developing leaves. This indicates that leaves are not only important sources for IAA synthesis, but that normal leaf expansion depends on rigorous control of IAA homeostasis.

Arabidopsis↗
PubMed1994

Mutational effects of space flight on Zea mays seeds.

The growth and development of more than 500 Zea mays seeds flown on LDEF were studied. Somatic mutations, including white-yellow stripes on leaves, dwarfing, change of leaf sheath color or seedling color were observed in plants developed from these seeds. When the frequency of white-yellow formation was used as the endpoint and compared with data from ground based studies, the dose to which maize seeds might be exposed during the flight was estimated to be equivalent to 635 cGy of gamma rays. Seeds from one particular holder gave a high mutation frequency and a wide mutation spectrum. White-yellow stripes on leaves were also found in some of the inbred progenies from plants displayed somatic mutation. Electron microscopy studies showed that the damage of chloroplast development in the white-yellow stripe on leaves was similar between seeds flown on LDEF and that irradiated by accelerated heavy ions on ground.

Chloroplasts↗
PubMed2005

Laser capture microdissection for the analysis of gene expression during embryogenesis of Arabidopsis.

It is during embryogenesis that the body plan of the developing plant is established. Analysis of gene expression during embryogenesis has been limited due to the technical difficulty of accessing the developing embryo. Here we demonstrate that laser capture microdissection can be applied to the analysis of embryogenesis. We show how this technique can be used in concert with DNA microarray for the large-scale analysis of gene expression in apical and basal domains of the globular-stage and heart-stage embryo, respectively, when critical events of polarity, symmetry and biochemical differentiation are established. This high resolution spatial analysis shows that up to approximately 65% of the genome is expressed in the developing embryo, and that differential expression of a number of gene classes can be detected. We discuss the validity of this approach for the functional analysis of both published and previously uncharacterized essential genes.

Arabidopsis↗
PubMed2006

Mitochondrial metabolism in developing embryos of Brassica napus.

The metabolism of developing plant seeds is directed toward transforming primary assimilatory products (sugars and amino acids) into seed storage compounds. To understand the role of mitochondria in this metabolism, metabolic fluxes were determined in developing embryos of Brassica napus. After labeling with [1,2-(13)C2]glucose + [U-(13)C6]glucose, [U-(13)C3]alanine, [U-(13)C5]glutamine, [(15)N]alanine, (amino)-[(15)N]glutamine, or (amide)-[(15)N]glutamine, the resulting labeling patterns in protein amino acids and in fatty acids were analyzed by gas chromatography-mass spectrometry. Fluxes through mitochondrial metabolism were quantified using a steady state flux model. Labeling information from experiments using different labeled substrates was essential for model validation and reliable flux estimation. The resulting flux map shows that mitochondrial metabolism in these developing seeds is very different from that in either heterotrophic or autotrophic plant tissues or in most other organisms: (i) flux around the tricarboxylic acid cycle is absent and the small fluxes through oxidative reactions in the mitochondrion can generate (via oxidative phosphorylation) at most 22% of the ATP needed for biosynthesis; (ii) isocitrate dehydrogenase is reversible in vivo; (iii) about 40% of mitochondrial pyruvate is produced by malic enzyme rather than being imported from the cytosol; (iv) mitochondrial flux is largely devoted to providing precursors for cytosolic fatty acid elongation; and (v) the uptake of amino acids rather than anaplerosis via PEP carboxylase determines carbon flow into storage proteins.

Acetyl Coenzyme A↗
PubMed2006

Construction of an infectious cDNA clone of Ribgrass mosaic virus Shanghai isolate and its modification to express an epitope of Mycobacterium tuberculosis.

Infectious cDNA clones of the Shanghai isolate of Ribgrass mosaic virus (RMV) were produced by joining four overlapping cDNA fragments and also in a single step by long template PCR. After inoculation of Nicotiana glutinosa with either RNA transcripts or the cDNA under the control of the CaMV 35S promoter, plants developed typical symptoms, and viral coat protein could be detected in them by Western blot analysis. However, compared to plants inoculated with purified viral RNA, lesions were fewer and appeared more slowly. An epitope of the Mycobacterium tuberculosis 31-kDa protein was inserted at the C-terminus of the viral coat protein by PCR using two overlapping fragments. The modified clone was also infectious and the foreign epitope could be detected serologically in the electron microscope and by Western blot analysis. The results demonstrate the potential of RMV as a viral gene vector.

Antigens, Bacterial↗
PubMed2000

Differential expression of genes encoding cell wall proteins in vascular tissues from vertical and bent loblolly pine trees.

Differential expression of cell wall proteins during plant development and in response to biotic or abiotic stress suggests that these proteins may contribute in different ways to plant cell wall architecture. Because the wood of loblolly pine (Pinus taeda L.) is highly specialized in the formation of secondary cell walls, it is an ideal tissue for studying these proteins. The cDNAs coding for six novel cell wall associated proteins, as well as a homologue for a phytocyanin, were identified and characterized from differentiating xylem of loblolly pine. Three of these cDNAs encoded new putative loblolly pine arabinogalactan proteins, based on their structural similarity to classical arabinogalactan proteins (AGPs). In addition, one clone was related to the proline-rich protein group and the other two to the glycine-rich protein group and the mussel adhesive protein. Relative expression of these genes was examined in different tissues and organs from normal trees (needles, phloem and vertical wood), the underside of bent trees (compression wood) and the lateral sides of the bent stems (side wood). All clones, except one, were highly expressed in vascular tissues with noticeable differences among the three types of wood. Their relationships and diversity provide the first insights into concerted expression and related function for this important group of proteins in cell wall formation of wood.

Journal Article↗
PubMed1998

Phytochromes and photomorphogenesis in Arabidopsis.

Plants have evolved exquisite sensory systems for monitoring their light environment. The intensity, quality, direction and duration of light are continuously monitored by the plant and the information gained is used to modulate all aspects of plant development. Several classes of distinct photoreceptors, sensitive to different regions of the light spectrum, mediate the developmental responses of plants to light signals. The red-far-red light-absorbing, reversibly photochromic phytochromes are perhaps the best characterized of these. Higher plants possess a family of phytochromes, the apoproteins of which are encoded by a small, divergent gene family. Arabidopsis has five apophytochrome-encoding genes, PHYA-PHYE. Different phytochromes have discrete biochemical and physiological properties, are differentially expressed and are involved in the perception of different light signals. Photoreceptor and signal transduction mutants of Arabidopsis are proving to be valuable tools in the molecular dissection of photomorphogenesis. Mutants deficient in four of the five phytochromes have now been isolated. Their analysis indicates considerable overlap in the physiological functions of different phytochromes. In addition, mutants defining components acting downstream of the phytochromes have provided evidence that different members of the family use different signalling pathways.

Arabidopsis↗