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PubMed2004

Production of intergeneric somatic hybrids between round kumquat (Fortunella japonica Swingle) and 'Morita navel' orange (Citrus sinensis Osbeck).

Intergeneric somatic hybrids between embryogenic callus-derived protoplasts of round kumquat (Fortunella japonica Swingle) and 'Morita navel' orange (Citrus sinensis Osbeck) were produced by electrofusion. Among the eight different fusion strains obtained, six showed normal morphology, whereas the remaining two showed malformation. All the regenerated plants were intermediate in leaf morphology and had thick and round leaves, which are typical characteristics of polyploids. Ploidy analyses by flow cytometry and chromosome counting in root-tip cells revealed that these plants are amphidiploid (2n=4x=36). Hybridity of the fusion products was confirmed by random amplified polymorphic DNA and cleaved amplified polymorphic sequence (CAPS) analyses. Furthermore, analyses of chloroplast (cp) and mitochondrial (mt) DNA by CAPS showed that these somatic hybrids contained cp- and mt-DNA of round kumquat without recombination in the regions analyzed.

Chimera↗
PubMed1996

The Arabidopsis thaliana ACT4/ACT12 actin gene subclass is strongly expressed throughout pollen development.

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 beta-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.

Actins↗
PubMed1999

A maize glycine-rich protein is synthesized in the lateral root cap and accumulates in the mucilage.

The root cap functions in the perception of gravity, the protection of the root apical meristem, and facilitation of the passage of roots through the soil, but the genes involved in these functions are poorly understood. Here we report the isolation of a root-specific gene from the cap of maize (Zea mays L.) primary root by cDNA subtraction and differential screening. The gene zmGRP4 (Z. mays glycine rich protein 4) encodes a member of the glycine-rich proteins with a putative signal peptide at the amino terminus. The deduced molecular mass of mature zmGRP4 is 14.4 kD. In situ-hybridization analysis has shown zmGRP4 to be strongly expressed in the lateral root cap and weakly expressed in the root epidermis. A polyclonal antibody raised against recombinant zmGRP4 detected a protein of 36 kD in the insoluble protein fraction extracted from the root tip and the root proper, indicating posttranslational modification(s) of zmGRP4. Immunohistochemical analysis showed the accumulation of zmGRP4 in the mucilage that covers the root tip. These results indicate that lateral root-cap cells secrete modified zmGRP4 into the mucilage to which the protein may contribute to its characteristic physical properties.

Amino Acid Sequence↗
PubMed2006

Time-lapse analysis of stem-cell divisions in the Arabidopsis thaliana root meristem.

In the Arabidopsis root, asymmetric stem-cell divisions produce daughters that form the different root cell types. Here we report the establishment of a confocal tracking system that allows the analysis of numbers and orientations of cell divisions in root stem cells. The system provides direct evidence that stem cells have lower division rates than cells in the proximal meristem. It also allows tracking of cell division timing, which we have used to analyse the synchronization of root cap divisions. Finally, it gives new insights into lateral root cap formation: epidermal stem-cell daughters can rotate the orientation of the division plane like the stem cell.

Arabidopsis↗
PubMed2004

Transcriptional regulation and functional properties of Arabidopsis Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants.

Phosphate mobilization into the plant is a complex process requiring numerous transporters for absorption and translocation of this major nutrient. In the genome of Arabidopsis thaliana, nine closely related high affinity phosphate transporters have been identified but their specific roles remain unclear. Here we report the molecular, histological and physiological characterization of Arabidopsis pht1;4 high affinity phosphate transporter mutants. Using GUS-gene trap and in situ hybridization, Pht1;4 was found mainly expressed in inorganic phosphate (Pi) limiting medium in roots, primarily in the epidermis, the cortex and the root cap. In addition to this, expression was also observed at the lateral root branch points on the primary root and in the stele of lateral roots, suggesting a role of Pht1;4 in phosphate absorption and translocation from the growth medium to the different parts of the plant. Pi-starved pht1;4 plantlets exhibited a strong reduction of phosphate uptake capacity (40). This phenotype appears only related to the pht1;4 mutation as there were no obvious changes in the expression of other Pht1 family members in the mutants background. However, after 10 days of growth on phosphate deficient or sufficient medium, the Pi content in the mutants was not significantly different from that of the corresponding wild type controls. Furthermore, the mutants did not display any obvious growth defects or visible phenotypes when grown on a low phosphate containing medium. The work described here offers a first step in the complex genetic dissection of the phosphate transport system in planta.

Arabidopsis↗
PubMed1984

Inhibition of polar calcium movement and gravitropism in roots treated with auxin-transport inhibitors.

Primary roots of maize (Zea mays L.) and pea (Pisum sativum L.) exhibit strong positive gravitropism. In both species, gravistimulation induces polar movement of calcium across the root tip from the upper side to the lower side. Roots of onion (Allium cepa L.) are not responsive to gravity and gravistimulation induces little or no polar movement of calcium across the root tip. Treatment of maize or pea roots with inhibitors of auxin transport (morphactin, naphthylphthalamic acid, 2,3,5-triiodobenzoic acid) prevents both gravitropism and gravity-induced polar movement of calcium across the root tip. The results indicate that calcium movement and auxin movement are closely linked in roots and that gravity-induced redistribution of calcium across the root cap may play an important role in the development of gravitropic curvature.

Allium↗
PubMed1983

The state of gravity sensors and peculiarities of plant growth during different gravitational loads.

In the primary roots of lettuce shoots grown under altered gravitational conditions--180 degrees inversion on the centrifuged clinostat, horizontal clinostat and in dynamic weightlessness--localization of the cellular organelles, cell morphology and peculiarities of growth have been studied. Significant changes took place in the localization of amyloplasts on the horizontal clinostat. The changes of amyloplast position in the cap cells on the horizontal clinostat and under weightlessness are similar. A change of the normal shoot position (180 degrees inversion and horizontal clinostat) causes an inhibition of growth. Weightlessness increases the length of axial organs and cells in the zone of elongation, but decreases the nitotic index in comparison to the centrifuged control. The anlysis of the formation of generative organs has been carried out for Arabidopsis plants grown on board the orbital station Salyut-6. The ability of plants to undergo vegetative growth and to pass through early phases of generative development under weightlessness was confirmed.

Arabidopsis↗
PubMed2003

A xylogalacturonan epitope is specifically associated with plant cell detachment.

A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitope is restricted to loosely attached inner parenchyma cells at the inner face of the pea testa and does not occur in other cells of the testa. Elsewhere in the pea seedling, the LM8 epitope was found only in association with root cap cell development at the root apex. Furthermore, the LM8 epitope is specifically associated with root cap cells in a range of angiosperm species. In embryogenic carrot suspension cell cultures the epitope is abundant at the surface of cell walls of loosely attached cells in both induced and non-induced cultures. The LM8 epitope is the first cell wall epitope to be identified that is specifically associated with a plant cell separation process that results in complete cell detachment.

Antibodies, Monoclonal↗
PubMed2003

Ionic signaling in plant gravity and touch responses.

Plant roots are optimized to exploit resources from the soil and as each root explores this environment it will encounter a range of biotic and abiotic stimuli to which it must respond. Therefore, each root must possess a sensory array capable of monitoring and integrating these diverse stimuli to direct the appropriate growth response. Touch and gravity represent two of the biophysical stimuli that plants must integrate. As sensing both of these signals requires mechano-transduction of biophysical forces to biochemical signaling events, it is likely that they share signal transduction elements. These common signaling components may allow for cross-talk and so integration of thigmotropic and gravitropic responses. Indeed, signal transduction events in both plant touch and gravity sensing are thought to include Ca(2+)- and pH-dependent events. Additionally, it seems clear that the systems responsible for root touch and gravity response interact to generate an integrated growth response. Thus, primary and lateral roots of Arabidopsis respond to mechanical stimuli by eliciting tropic growth that is likely part of a growth strategy employed by the root to circumvent obstacles in the soil. Also, the mechano-signaling induced by encountering an obstacle apparently down-regulates the graviperception machinery to allow this kind of avoidance response. The challenge for future research will be to define how the cellular signaling events in the root cap facilitate this signal integration and growth regulation. In addition, whether other stimuli are likewise integrated with the graviresponse via signal transduction system cross-talk is an important question that remains to be answered.

Arabidopsis↗
PubMed1995

Novel fixation of plant tissue, staining through paraffin with alcian blue and hematoxylin, and improved slide preparation.

Onion (Allium cepa) root tips were fixed in a proprietary solution without aldehyde, toxic metals or acetic acid. Fixed specimens were embedded in paraffin, sectioned on a rotary microtome and mounted on detergent-washed slides without adhesive. Slides with ribbon segments affixed were immersed in 0.2% aqueous alcian blue 8GX in screw-capped Coplin jars in a water bath at 50 C for 1 hr. Excess alcian blue was rinsed off under cold running tap water and the slides were immersed in quick-mixed hematoxylin at room temperature for 15 min. Stained slides were deparaffinized, rinsed with isopropanol, air dried, and coverslips were affixed with resin. Thus, the traditional paraffin microtechnique has been modified at all steps from fixation to finishing slides with coverslips.

Alcian Blue↗
PubMed1991

Genetic and molecular analyses of picA, a plant-inducible locus on the Agrobacterium tumefaciens chromosome.

picA is an Agrobacterium tumefaciens chromosomal locus, identified by Mu d11681 mutagenesis, that is inducible by certain acidic polysaccharides found in carrot root extract. Cloning and genetic analysis of a picA::lacZ fusion defined a region of the picA promoter that is responsible for the induction of this locus. Furthermore, we identified a possible negative regulator of picA expression upstream of the picA locus. This sequence, denoted pgl, has extensive homology to polygalacturonase genes from several organisms and inhibited the induction of the picA promoter when present in multiple copies in A. tumefaciens. DNA sequence analysis indicated at least two long open reading frames (ORFs) in the picA region. S1 nuclease mapping was used to identify the transcription initiation site of picA. Mutation of ORF1, but not ORF2, of the picA locus was responsible for an increased aggregation of A. tumefaciens, forming "ropes" in the presence of pea root cap cells. In addition, a potato tuber disk virulence assay indicated that a preinduced picA mutant was more virulent than was the wild-type control, a further indication that the picA locus regulates the surface properties of the bacterium in the presence of plant cells or plant cell extracts.

Amino Acid Sequence↗
PubMed2004

Root border cell development is a temperature-insensitive and Al-sensitive process in barley.

In vivo and in vitro experiments showed that border cell (BC) survival was dependent on root tip mucigel in barley (Hordeum vulgare L. cv. Hang 981). In aeroponic culture, BC development was an induced process in barley, whereas in hydroponic culture, it was a kinetic equilibrium process during which 300-400 BCs were released into water daily. The response of root elongation to temperatures (10-35 degrees C) was very sensitive but temperature changes had no great effect on barley BC development. At 35 degrees C, the root elongation ceased whereas BC production still continued, indicating that the two processes might be regulated independently under high temperature (35 degrees C) stress. Fifty microM Al could inhibit significantly BC development by inhibiting pectin methylesterase activity in the root cap of cv. 2000-2 (Al-sensitive) and cv. Humai 16 (Al-tolerant), but 20 microM Al could not block BC development in cv. Humai 16. BCs and their mucigel of barley had a limited role in the protection of Al-induced inhibition of root elongation, but played a significant role in the prevention of Al from diffusing into the meristems of the root tip and the root cap. Together, these results suggested that BC development was a temperature-insensitive but Al-sensitive process, and that BCs and their mucigel played an important role in the protection of root tip and root cap meristems from Al toxicity.

Aluminum↗
PubMed2025

Dynamic Rhizodeposition in the Woody Perennial Populus trichocarpa.

Plants undergo physiological and metabolic changes that release specific molecules into the surrounding soil, a process collectively known as rhizodeposition. These compounds play crucial roles in plant-microbe-soil interactions, such as supporting plant development and resilience in changing environments. Under nutrient-limited conditions, these plant-derived compounds modify the rhizosphere environment, mobilizing otherwise inaccessible nutrients and recruiting stress-adaptive microbial communities that support stress resilience. Currently, the chemical diversity of rhizodeposition has yet to be fully realized but is expected to be a complex mixture that includes soluble organic compounds excreted from root cells, along with products of root cell turnover, sloughed-off root cap and border cells, and mucilage. Here, we developed a methodological and conceptual framework for an in-depth measurement of rhizodeposition through critical advancements in untargeted metabolomics. This approach provided foundational insights into the dynamic changes in rhizodeposition for the woody perennial Populus trichocarpa and rhizodeposit profiles varying by genotype, time, location, and environment. More broadly, this study provides a framework that will help formulate the next steps to effectively study rhizodeposition.

Populus↗
PubMed2003

The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway.

The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

Amino Acid Sequence↗
PubMed2005

Altered microtubule dynamics by expression of modified alpha-tubulin protein causes right-handed helical growth in transgenic Arabidopsis plants.

The proper organization of cortical microtubule arrays is essential for anisotropic growth in plants but how distinct array patterns are formed is not understood. Here, we report a relationship between microtubule dynamics and array organization using transgenic plants expressing modified tubulins. When green fluorescent protein (GFP) or a hemaglutinin epitope tag was fused to the N-terminus of tubulins and expressed in Arabidopsis plants, these tubulins were incorporated into microtubules along with endogenous tubulins. Plants expressing the modified beta-tubulins were phenotypically normal and possessed transversely oriented cortical arrays in the epidermal cells of the root elongation zone; however, the expression of modified alpha-tubulins caused right-handed helical growth, increased trichome branching, and a shallow left-handed (S-form) helical array organization. In cells expressing the modified alpha-tubulins, microtubule dynamicity was suppressed and polymerization was promoted, and GFP-EB1 (End Binding 1) labeled larger regions of the microtubule end more frequently, when compared with control cells. We propose that the N-terminal appendage introduced into alpha-tubulin inhibits GTP hydrolysis, thus producing polymerization-prone microtubules with an extended GTP cap. Consistent with this interpretation, plants expressing an alpha-tubulin mutated in the GTPase-activating domain exhibited similar microtubule properties, with regard to dynamics and the localization of GFP-EB1, and showed right-handed helical growth.

Arabidopsis↗
PubMed2004

Role of cytokinin in the regulation of root gravitropism.

The models explaining root gravitropism propose that the growth response of plants to gravity is regulated by asymmetric distribution of auxin (indole-3-acetic acid, IAA). Since cytokinin has a negative regulatory role in root growth, we suspected that it might function as an inhibitor of tropic root elongation during gravity response. Therefore, we examined the free-bioactive-cytokinin-dependent ARR5::GUS expression pattern in root tips of transformants of Arabidopsis thaliana (L.) Heynh., visualized high cytokinin concentrations in the root cap with specific monoclonal antibodies, and complemented the analyses by external application of cytokinin. Our findings show that mainly the statocytes of the cap produce cytokinin, which may contribute to the regulation of root gravitropism. The homogenous symmetric expression of the cytokinin-responsive promoter in vertical root caps rapidly changed within less than 30 min of gravistimulation into an asymmetrical activation pattern, visualized as a lateral, distinctly stained, concentrated spot on the new lower root side of the cap cells. This asymmetric cytokinin distribution obviously caused initiation of a downward curvature near the root apex during the early rapid phase of gravity response, by inhibiting elongation at the lower side and promoting growth at the upper side of the distal elongation zone closely behind the root cap. Exogenous cytokinin applied to vertical roots induced root bending towards the application site, confirming the suspected inhibitory effect of cytokinin in root gravitropism. Our results suggest that the early root graviresponse is controlled by cytokinin. We conclude that both cytokinin and auxin are key hormones that regulate root gravitropism.

Arabidopsis↗
PubMed1999

Plastid sedimentation kinetics in roots of wild-type and starch-deficient mutants of Arabidopsis.

Sedimentation and movement of plastids in columella cells of the root cap were measured in seedlings of wild-type, a reduced starch mutant, and a starchless mutant of Arabidopsis. To assay for sedimentation, we used both linear measurements and the change of angle from the cell center as indices in vertical and reoriented plants with the aid of computer-assisted image analysis. Seedlings were fixed at short periods after reorientation, and plastid sedimentation correlated with starch content in the three strains of Arabidopsis. Amyloplasts of wild-type seedlings showed the greatest sedimentation, whereas plastids of the starchless mutant showed no significant sedimentation in the vertically grown and reoriented seedlings. Because previous research has shown that a full complement of starch is needed for full gravitropic sensitivity, this study correlates increased sensitivity with plastid sedimentation. However, although plastid sedimentation contributed to gravisensitivity, it was not required, because the gravitropic starchless mutant had plastids that did not sediment. This is the first study, to our knowledge, to measure plastid sedimentation in Arabidopsis roots after reorientation of seedlings. Taken together, the results of this study are consistent with the classic plastid-based and protoplast-based models of graviperception and suggest that multiple systems of perception exist in plant cells.

Arabidopsis↗
PubMed1987

Effects of microfilament disrupters on microfilament distribution and morphology in maize root cells.

Maize root tip cells were examined for the distribution of actin microfilaments in various cell types and to determine the effects of microfilament disrupters. Fluorescence microscopy on fixed, stabilized, squashed cells using the F-actin specific probe, rhodamine-labelled phalloidin, allowed for a three-dimensional visualization of actin microfilaments. Microfilaments were observed as long, meandering structures in root cap cells and meristematic cells, while those in immature vascular parenchyma were abundant in the thin band of cytoplasm and were long and less curved. By modifying standard electron microscopic fixation procedures, microfilaments in plant cells could be easily detected in all cell types. Treatment with cytochalasin B, cytochalasin D and lead acetate, compounds that interfere with microfilament related processes, re-organized the microfilaments into abnormal crossed and highly condensed masses. All the treatments affected not only the microfilaments but also the accumulation of secretory vesicles. The vivid demonstration of the effects of all of these microfilament disrupters on the number and size of Golgi vesicles indicates that these vesicles may depend on microfilaments for intracellular movement.

Actin Cytoskeleton↗