Biomedical subjects

Huazhong Shi

Publications and source records attributed to Huazhong Shi.

12 recordsLinked to original sources

PubMed2006

Salt stress affects cortical microtubule organization and helical growth in Arabidopsis.

Cortical microtubule arrays are critical in determining the growth axis of diffusely growing plant cells, and various environmental and physiological factors are known to affect the array organization. Microtubule organization is partly disrupted in the spiral1 mutant of Arabidopsis thaliana, which displays a right-handed helical growth phenotype in rapidly elongating epidermal cells. We show here that mutations in the plasma membrane Na(+)/H(+) antiporter SOS1 and its regulatory kinase SOS2 efficiently suppressed both microtubule disruption and helical growth phenotypes of spiral1, and that sos1 and sos2 roots in the absence of salt stress exhibited altered helical growth response to microtubule-interacting drugs at low doses. Salt stress also altered root growth response to the drugs in wild-type roots. Suppression of helical growth appeared to be specific to spiral1 since other helical growth mutants were not rescued. The effects of sos1 in suppressing spiral1 defects and in causing abnormal drug responses were nullified in the presence of the hkt1 Na(+) influx carrier mutation in roots but not in hypocotyls. These results suggest that cytoplasmic salt imbalance caused by insufficient SOS1 activity compromises cortical microtubule functions in which microtubule-localized SPIRAL1 is specifically involved.

Arabidopsis↗

PubMed2006

RNA extraction.

RNA extraction is a routine technique in a molecular biology lab. High quality of RNA extracted from plants is a prerequisite to succeeding in subsequent experiments such as reverse transcription-polymerase chain reaction, Northern hybridization, cDNA library construction, and microarray analysis. Two methods for RNA extraction in Arabidopsis, small-scale and large-scale, are described here. RNA isolated by using the small-scale method can be applied in most downstream applications. The large-scale RNA preparation is recommended to extract RNA for Northern hybridization only.

Arabidopsis↗

PubMed2004

Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles.

Salt cress (Thellungiella halophila) is a small winter annual crucifer with a short life cycle. It has a small genome (about 2 x Arabidopsis) with high sequence identity (average 92%) with Arabidopsis, and can be genetically transformed by the simple floral dip procedure. It is capable of copious seed production. Salt cress is an extremophile native to harsh environments and can reproduce after exposure to extreme salinity (500 mm NaCl) or cold to -15 degrees C. It is a typical halophyte that accumulates NaCl at controlled rates and also dramatic levels of Pro (>150 mm) during exposure to high salinity. Stomata of salt cress are distributed on the leaf surface at higher density, but are less open than the stomata of Arabidopsis and respond to salt stress by closing more tightly. Leaves of salt cress are more succulent-like, have a second layer of palisade mesophyll cells, and are frequently shed during extreme salt stress. Roots of salt cress develop both an extra endodermis and cortex cell layer compared to Arabidopsis. Salt cress, although salt and cold tolerant, is not exceptionally tolerant of soil desiccation. We have isolated several ethyl methanesulfonate mutants of salt cress that have reduced salinity tolerance, which provide evidence that salt tolerance in this halophyte can be significantly affected by individual genetic loci. Analysis of salt cress expressed sequence tags provides evidence for the presence of paralogs, missing in the Arabidopsis genome, and for genes with abiotic stress-relevant functions. Hybridizations of salt cress RNA targets to an Arabidopsis whole-genome oligonucleotide array indicate that commonly stress-associated transcripts are expressed at a noticeably higher level in unstressed salt cress plants and are induced rapidly under stress. Efficient transformation of salt cress allows for simple gene exchange between Arabidopsis and salt cress. In addition, the generation of T-DNA-tagged mutant collections of salt cress, already in progress, will open the door to a new era of forward and reverse genetic studies of extremophile plant biology.

Abscisic Acid↗

PubMed2004

An Arabidopsis homeodomain transcription factor gene, HOS9, mediates cold tolerance through a CBF-independent pathway.

To investigate essential components mediating stress signaling in plants, we initiated a large-scale stress response screen using Arabidopsis plants carrying the firefly luciferase reporter gene under the control of the stress-responsive RD29A promoter. Here we report the identification and characterization of a mutant, hos9-1 (for high expression of osmotically responsive genes), in which the reporter construct was hyperactivated by low temperature, but not by abscisic acid or salinity stress. The mutants grow more slowly, and flower later, than do wild-type plants and are more sensitive to freezing, both before and after acclimation, than the wild-type plants. The HOS9 gene encodes a putative homeodomain transcription factor that is localized to the nucleus. HOS9 is constitutively expressed and not further induced by cold stress. Cold treatment increased the level of transcripts of the endogenous RD29A, and some other stress-responsive genes, to a higher level in hos9-1 than in wild-type plants. However, the C repeat/dehydration responsive element-binding factor (CBF) transcription factor genes that mediate a part of cold acclimation in Arabidopsis did not have their response to cold altered by the hos9-1 mutation. Correspondingly, microarray analysis showed that none of the genes affected by the hos9-1 mutation are controlled by the CBF family. Together, these results suggest that HOS9 is important for plant growth and development, and for a part of freezing tolerance, by affecting the activity of genes independent of the CBF pathway.

Abscisic Acid↗

PubMed2003

Topological analysis of a plant vacuolar Na+/H+ antiporter reveals a luminal C terminus that regulates antiporter cation selectivity.

We conducted an analysis of the topology of AtNHX1, an Arabidopsis thaliana vacuolar Na+/H+ antiporter. Several hydrophilic regions of the antiporter were tagged with a hemagglutinin epitope, and protease protection assays were conducted to determine the membrane topology of the antiporter by using yeast as a heterologous expression system. The overall structure of AtNHX1 is distinct from the human Na+/H+ antiporter NHE1 or any known Na+/H+ antiporter. It is comprised of nine transmembrane domains and a hydrophilic C-terminal domain. Three hydrophobic regions do not appear to span the tonoplast membrane, yet appear to be membrane associated. Our results also indicate that, whereas the N terminus of AtNHX1 is facing the cytosol, almost the entire C-terminal hydrophilic region resides in the vacuolar lumen. Deletion of the hydrophilic C terminus resulted in a dramatic increase in the relative rate of Na+/H+ transport. The ratio of Na+/K+ transport was twice that of the unmodified AtNHX1. This altered ratio resulted from a relatively small decrease in K+/H+ transport with a large increase in Na+/H+ transport. The vacuolar localization of the C terminus of the AtNHX1, taken together with the regulation of the antiporter selectivity by its C terminus, demonstrates the existence of luminal vacuolar regulatory mechanisms of the antiporter activity.

Arabidopsis↗

PubMed2003

The Arabidopsis SOS5 locus encodes a putative cell surface adhesion protein and is required for normal cell expansion.

Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein-like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf.

Amino Acid Sequence↗

PubMed2002

Overexpression of a plasma membrane Na+/H+ antiporter gene improves salt tolerance in Arabidopsis thaliana.

High concentrations of Na+ in saline soils inhibit plant growth and reduce agricultural productivity. We report here that CaMV 35S promoter driven overexpression of the Arabidopsis thaliana SOS1 gene, which encodes a plasma membrane Na+/H+ antiporter, improves plant salt tolerance in A. thaliana. Transgenic plants showed substantial upregulation of SOS1 transcript levels upon NaCl treatment, suggesting post-transcriptional control of SOS1 transcript accumulation. In response to NaCl treatment, transgenic plants overexpressing SOS1 accumulated less Na+ in the xylem transpirational stream and in the shoot. Undifferentiated callus cultures regenerated from the transgenic plants were also more tolerant of salt stress, which was correlated with reduced Na+ content in the transgenic cells. These results show that improved salt tolerance could be achieved by limiting Na+ accumulation in plant cells.

Arabidopsis↗

PubMed2002

Reconstitution in yeast of the Arabidopsis SOS signaling pathway for Na+ homeostasis.

The Arabidopsis thaliana SOS1 protein is a putative Na+/H+ antiporter that functions in Na+ extrusion and is essential for the NaCl tolerance of plants. sos1 mutant plants share phenotypic similarities with mutants lacking the protein kinase SOS2 and the Ca2+ sensor SOS3. To investigate whether the three SOS proteins function in the same response pathway, we have reconstituted the SOS system in yeast cells. Expression of SOS1 improved the Na+ tolerance of yeast mutants lacking endogenous Na+ transporters. Coexpression of SOS2 and SOS3 dramatically increased SOS1-dependent Na+ tolerance, whereas SOS2 or SOS3 individually had no effect. The SOS2/SOS3 kinase complex promoted the phosphorylation of SOS1. A constitutively active form of SOS2 phosphorylated SOS1 in vitro independently of SOS3, but could not fully substitute for the SOS2/SOS3 kinase complex for activation of SOS1 in vivo. Further, we show that SOS3 recruits SOS2 to the plasma membrane. Although sos1 mutant plants display defective K+ uptake at low external concentrations, neither the unmodified nor the SOS2/SOS3-activated SOS1 protein showed K+ transport capacity in vivo, suggesting that the role of SOS1 on K+ uptake is indirect. Our results provide an example of functional reconstitution of a plant response pathway in a heterologous system and demonstrate that the SOS1 ion transporter, the SOS2 protein kinase, and its associated Ca2+ sensor SOS3 constitute a functional module. We propose a model in which SOS3 activates and directs SOS2 to the plasma membrane for the stimulatory phosphorylation of the Na+ transporter SOS1.

Arabidopsis↗

PubMed2002

Regulation of expression of the vacuolar Na+/H+ antiporter gene AtNHX1 by salt stress and abscisic acid.

The Arabidopsis thaliana AtNHX1 gene encodes a vacuolar Na+/H+ antiporter that is important in salt tolerance. We report here the tissue distribution and regulation of AtNHX1 expression by salt stress and abscisic acid (ABA). The steady-state level of AtNHX1 transcript was up-regulated by treatment with NaCl, KCl or ABA. AtNHX1 promoter-GUS analysis in transgenic Arabidopsis showed that AtNHX1 was expressed in all tissues except the root tip. Strong GUS expression was detected in guard cells, suggesting that AtNHX1 may play a role in pH regulation and/or K+ homeostasis in the specialized cells. AtNHX1 promoter activity was substantially up-regulated by NaCl, KCl or ABA, demonstrating that salt and ABA regulation of AtNHX1 expression occurs at the transcriptional level. Strong induction of GUS activity in root hair cells was observed, which suggests a role of AtNHX1 in storing Na+ in the enlarged vacuoles in root hair cells. The up-regulation of AtNHX1 transcript levels by NaCl was reduced in abi1-1, aba2-1 and aba3-1, but not in abi2-1, sos1, sos2 or sos3 mutants. ABA-induced AtNHX1 expression was also decreased in abi1-1 but not in abi2-1. These results suggest that salt stress up-regulates AtNHX1 expression transcriptionally and the up-regulation is partially dependent on ABA biosynthesis and ABA signaling through ABI1.

Abscisic Acid↗

PubMed2002

SOS4, a pyridoxal kinase gene, is required for root hair development in Arabidopsis.

Root hair development in plants is controlled by many genetic, hormonal, and environmental factors. A number of genes have been shown to be important for root hair formation. Arabidopsis salt overly sensitive 4 mutants were originally identified by screening for NaCl-hypersensitive growth. The SOS4 (Salt Overly Sensitive 4) gene was recently isolated by map-based cloning and shown to encode a pyridoxal (PL) kinase involved in the production of PL-5-phosphate, which is an important cofactor for various enzymes and a ligand for certain ion transporters. The root growth of sos4 mutants is slower than that of the wild type. Microscopic observations revealed that sos4 mutants do not have root hairs in the maturation zone. The sos4 mutations block the initiation of most root hairs, and impair the tip growth of those that are initiated. The root hairless phenotype of sos4 mutants was complemented by the wild-type SOS4 gene. SOS4 promoter-beta-glucuronidase analysis showed that SOS4 is expressed in the root hair and other hair-like structures. Consistent with SOS4 function as a PL kinase, in vitro application of pyridoxine and pyridoxamine, but not PL, partially rescued the root hair defect in sos4 mutants. 1-Aminocyclopropane-1-carboxylic acid and 2,4-dichlorophenoxyacetic acid treatments promoted root hair formation in both wild-type and sos4 plants, indicating that genetically SOS4 functions upstream of ethylene and auxin in root hair development. The possible role of SOS4 in ethylene and auxin biosynthesis is discussed.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2002

The putative plasma membrane Na(+)/H(+) antiporter SOS1 controls long-distance Na(+) transport in plants.

The salt tolerance locus SOS1 from Arabidopsis has been shown to encode a putative plasma membrane Na(+)/H(+) antiporter. In this study, we examined the tissue-specific pattern of gene expression as well as the Na(+) transport activity and subcellular localization of SOS1. When expressed in a yeast mutant deficient in endogenous Na(+) transporters, SOS1 was able to reduce Na(+) accumulation and improve salt tolerance of the mutant cells. Confocal imaging of a SOS1-green fluorescent protein fusion protein in transgenic Arabidopsis plants indicated that SOS1 is localized in the plasma membrane. Analysis of SOS1 promoter-beta-glucuronidase transgenic Arabidopsis plants revealed preferential expression of SOS1 in epidermal cells at the root tip and in parenchyma cells at the xylem/symplast boundary of roots, stems, and leaves. Under mild salt stress (25 mM NaCl), sos1 mutant shoot accumulated less Na(+) than did the wild-type shoot. However, under severe salt stress (100 mM NaCl), sos1 mutant plants accumulated more Na(+) than did the wild type. There also was greater Na(+) content in the xylem sap of sos1 mutant plants exposed to 100 mM NaCl. These results suggest that SOS1 is critical for controlling long-distance Na(+) transport from root to shoot. We present a model in which SOS1 functions in retrieving Na(+) from the xylem stream under severe salt stress, whereas under mild salt stress it may function in loading Na(+) into the xylem.

Arabidopsis↗

PubMed2002

The Arabidopsis salt overly sensitive 4 mutants uncover a critical role for vitamin B6 in plant salt tolerance.

Salt stress is a major environmental factor influencing plant growth and development. To identify salt tolerance determinants, a genetic screen for salt overly sensitive (sos) mutants was performed in Arabidopsis. We present here the characterization of sos4 mutants and the positional cloning of the SOS4 gene. sos4 mutant plants are hypersensitive to Na(+), K(+), and Li(+) ions. Under NaCl stress, sos4 plants accumulate more Na(+) and retain less K(+) compared with wild-type plants. SOS4 encodes a pyridoxal kinase that is involved in the biosynthesis of pyridoxal-5-phosphate, an active form of vitamin B6. The expression of SOS4 cDNAs complements an Escherichia coli mutant defective in pyridoxal kinase. Supplementation of pyridoxine but not pyridoxal in the growth medium can partially rescue the sos4 defect in salt tolerance. SOS4 is expressed ubiquitously in all plant tissues. As a result of alternative splicing, two transcripts are derived from the SOS4 gene, the relative abundance of which is modulated by development and environmental stresses. Besides being essential cofactors for numerous enzymes, as shown by pharmacological studies in animal cells, pyridoxal-5-phosphate and its derivatives are also ligands for P2X receptor ion channels. Our results demonstrate that pyridoxal kinase is a novel salt tolerance determinant important for the regulation of Na(+) and K(+) homeostasis in plants. We propose that pyridoxal-5-phosphate regulates Na(+) and K(+) homeostasis by modulating the activities of ion transporters.

Adaptation, Physiological↗