Keni Jiang

Quiescent center formation in maize roots is associated with an auxin-regulated oxidizing environment

Embedded within the meristem of all Angiosperm roots is a population of slowly dividing cells designated the quiescent center (QC). In maize roots the QC can constitute upwards of 800-1200 cells, most of which spend an extended period of time (180-200 hours) in the G1 phase of the cell cycle. How the QC forms and is maintained is not known. Here we report that cells of the QC are characterized by their highly oxidized status. Glutathione and ascorbic acid occur predominately in the oxidized forms in the QC. This is contrasted with the status of these redox intermediates in adjacent, rapidly dividing cells in the root meristem, in which the reduced forms of these two species are favored. Using a redox sensitive fluorescent dye we were able to visualize an overall oxidizing environment in the QC, and we also made comparisons with the adjacent, rapidly dividing cells in the root meristem. Altering the distribution of auxin and the location of the auxin maximum in the root tip activates the QC, and cells leave G1 and enter mitosis. Commencement of relatively more rapid cell division in the QC is preceded by changes in the overall redox status of the QC, which becomes less oxidizing. We discuss how the position of the auxin maximum may influence the redox status of the QC and thereby modulate the cell cycle.

Expression and Characterization of a Redox-Sensing Green Fluorescent Protein (Reduction-Oxidation-Sensitive Green Fluorescent Protein) in Arabidopsis

Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are −318 mV for the cytoplasm and −362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.

Redox regulation of root apical meristem organization: Connecting root development to its environment

Post-embryonic root growth relies on the proliferative activity of the root apical meristem (RAM), consisting, in part, of cells with juvenile characteristics (stem cells). It is generally, but erroneously held that the RAM indefinitely produces new cells throughout the lifespan of a plant, resulting in indeterminate root growth. On the contrary, convincing data, mainly from the lab of Thomas L. Rost, show in all species analyzed so far, including Arabidopsis, that RAM organization changes over time in parallel with both a cessation of the production of new cells, and a consequent reduction in root growth, even under optimal conditions. In addition, RAM organization evolved to become highly plastic and dynamic in response to environmental triggers (e.g. water and nutrient availability, pollutants). Under unfavourable conditions, the RAM is rapidly reorganized, and, as a result of the cessation of new cell production at the root tip, root growth is altered, and lateral root production is enhanced, thus providing the plant additional strategies to overcome the stress. It is now becoming increasingly clear that this environment-responsive developmental plasticity is linked to reactive oxygen/nitrogen species, antioxidants, and related enzymes, which form part of a complex signalling module specifically operating in the regulation of RAM functioning, in strict relationship with hormonal control of root development exerted by auxin, gibberellins and cytokinins. In turn, such redox/hormone crosstalk regulates gene expression.

Redox states of plastids and mitochondria differentially regulate intercellular transport via plasmodesmata

Recent studies suggest that intercellular transport via plasmodesmata (PD) is regulated by cellular redox state. Until now, this relationship has been unclear, as increased production of reactive oxygen species (ROS) has been associated with both increased and decreased intercellular transport via PD. Here, we show that silencing two genes that both increase transport via PD, INCREASED SIZE EXCLUSION LIMIT1 (ISE1) and ISE2, alters organelle redox state. Using redox-sensitive green fluorescent proteins targeted to the mitochondria or plastids, we show that, relative to wild-type leaves, plastids are more reduced in both ISE1- and ISE2-silenced leaves, whereas mitochondria are more oxidized in ISE1-silenced leaves. We further show that PD transport is positively regulated by ROS production in mitochondria following treatment with salicylhydroxamic acid but negatively regulated by an oxidative shift in both chloroplasts and mitochondria following treatment with paraquat. Thus, oxidative shifts in the mitochondrial redox state positively regulate intercellular transport in leaves, but oxidative shifts in the plastid redox state counteract this effect and negatively regulate intercellular transport. This proposed model reconciles previous contradictory evidence relating ROS production to PD transport and supports accumulating evidence that mitochondria and plastids are crucial regulators of PD function.

Transcription profile analyses identify genes and pathways central to root cap functions in maize.

Affymetrix GeneChips arrayed with about one-half (~23K) of the rice genes were used to profile gene transcription activity in three tissues comprising the maize root tip; the proximal meristem (PM), the quiescent center (QC), and the root cap (RC). Here we analyze the gene transcription profile of the RC, compared to both the PM and the QC, from three biological replicates. In the RC, a total of 669 genes were identified as being differentially upregulated, and 365 differentially downregulated. Real-time quantitative RT-PCR analysis was used to confirm upregulated genes in the RC. In addition, using the technique of laser microdissection (LMD) we localized upregulated gene expression to the lateral RC cells. Taken as a whole, transcription profile analyses revealed the upregulation in the maize RC of clusters of genes linked to major metabolic processes and pathways, including: (1) transport, both the export of carbohydrates and the uptake of nutrients; (2) sensing and responding to (often stressful) biotic and abiotic environmental stimuli; (3) integrating the responses of at least 3 major growth regulators (auxin, ethylene, jasmonic acid); (4) processing the large amount of carbohydrate transported into the RC. Although the profile data are derived using heterologous rice GeneChips, with about half of the total rice gene set, this study, nevertheless, provides a genomic scale characterization of the entire RC, and serves as a new platform from which to advance studies of the network of pathways operating in the maize RC.

Root Meristem Establishment and Maintenance: The Role of Auxin

Auxin has a central role in the establishment and elaboration of pattern in root meristems. Regulation of root development by auxin begins early in embryogenesis, perhaps even as early as the establishment of polarity in the zygote, and persists throughout the lifetime of a root. Auxin-regulated development depends on a balance of synthesis/import and metabolism/export/sequestration. The overall result of these processes is to establish a state of auxin homeostasis which we hypothesize is required for normal root meristem patterning and development.

Use of a redox-sensing GFP (c-roGFP1) for real-time monitoring of cytosol redox status in Arabidopsis thaliana water-stressed plants.

Using Arabidopsis plants transformed with a redox‐sensing green fluorescent protein targeted to the cytosol (c‐roGFP1), we have demonstrated, in real time, measurements of reversible changes of redox status in the cytosol of plants subjected to a gradual water‐stress, followed by re‐watering. Plants sensed water stress, and changed the redox potential of their cytosol to a more oxidized value after a gradually‐imposed water stress. Small variations in the cytosol redox potential and ascorbate (AA) values suggest that this parameter was tightly regulated. The re‐watering was paralleled by a return of water stress, redox potential and ascorbate to initial values, showing the reversibility of water stress and its consequences.

A fluorometer-based method for monitoring oxidation of redox-sensitive GFP (roGFP) during development and extended dark stress

Redox-sensitive GFP (roGFP) localized to different compartments has been shown to be suitable for determination of redox potentials in plants via imaging. Long-term measurements bring out the need for analyzing a large number of samples which are averaged over a large population of cells. Because this goal is too tedious to be achieved by confocal imaging, we have examined the possibility of using a fluorometer to monitor changes in roGFP localized to different subcellular compartments during development and dark-induced senescence. The degree of oxidations determined by a fluorometer for different probes was similar to values obtained by confocal image analysis. Comparison of young and old leaves indicated that in younger cells higher levels of H(2)O(2) were required to achieve full roGFP oxidation, a parameter which is necessary for calculation of the degree of oxidation of the probe and the actual redox potential. Therefore, it is necessary to carefully determine the H(2)O(2) concentration required to achieve full oxidation of the probe. In addition, there is an increase in autofluorescence during development and extended dark stress, which might interfere with the ability to detect changes in oxidation-reduction dependent fluorescence of roGFP. Nevertheless, it was possible to determine the full dynamic range between the oxidized and the reduced forms of the different probes in the various organelles until the third day of darkness and during plant development, thereby enabling further analysis of probe oxidation. Hence, fluorometer measurements of roGFP can be used for extended measurements enabling the processing of multiple samples. It is envisaged that this technology may be applicable to the analysis of redox changes in response to other stresses or to various mutants.

A Role for Mitochondria in the Establishment and Maintenance of the Maize Root Quiescent Center

Mitochondria in the oxidizing environment of the maize (Zea mays) root quiescent center (QC) are altered in function, but otherwise structurally normal. Compared to mitochondria in the adjacent, rapidly dividing cells of the proximal root tissues, mitochondria in the QC show marked reductions in the activities of tricarboxylic acid cycle enzymes. Pyruvate dehydrogenase activity was not detected in the QC. Use of several mitochondrial membrane potential (ΔΨm) sensing probes indicated a depolarization of the mitochondrial membrane in the QC, which suggests a reduction in the capacity of QC mitochondria to generate ATP and NADH. We postulate that modifications of mitochondrial function are central to the establishment and maintenance of the QC.

Measuring similarities between gene expression profiles through new data transformations

BackgroundClustering methods are widely used on gene expression data to categorize genes with similar expression profiles. Finding an appropriate (dis)similarity measure is critical to the analysis. In our study, we developed a new measure for clustering the genes when the key factor is the shape of the profile, and when the expression magnitude should also be accounted for in determining the gene relationship. This is achieved by modeling the shape and magnitude parameters separately in a gene expression profile, and then using the estimated shape and magnitude parameters to define a measure in a new feature space.ResultsWe explored several different transformation schemes to construct the feature spaces that include a space whose features are determined by the mutual differences of the original expression components, a space derived from a parametric covariance matrix, and the principal component space in traditional PCA analysis. The former two are the newly proposed and the latter is explored for comparison purposes. The new measures we defined in these feature spaces were employed in a K-means clustering procedure to perform analyses. Applying these algorithms to a simulation dataset, a developing mouse retina SAGE dataset, a small yeast sporulation cDNA dataset, and a maize root affymetrix microarray dataset, we found from the results that the algorithm associated with the first feature space, named TransChisq, showed clear advantages over other methods.ConclusionThe proposed TransChisq is very promising in capturing meaningful gene expression clusters. This study also demonstrates the importance of data transformations in defining an efficient distance measure. Our method should provide new insights in analyzing gene expression data. The clustering algorithms are available upon request.