Genetic mapping and comparative analysis of seven mutants related to seed fiber development in cotton

Mapping of genes that play major roles in cotton fiber development is an important step toward their cloning and manipulation, and provides a test of their relationships (if any) to agriculturally-important QTLs. Seven previously identified fiber mutants, four dominant (Li ₁, Li ₂, N ₁ and Fbl) and three recessive (n ₂, sma-4(h _a), and sma-4(fz)), were genetically mapped in six F₂ populations comprising 124 or more plants each. For those mutants previously assigned to chromosomes by using aneuploids or by linkage to other morphological markers, all map locations were concordant except n ₂, which mapped to the homoeolog of the chromosome previously reported. Three mutations with primary effects on fuzz fibers (N ₁, Fbl, n ₂) mapped near the likelihood peaks for QTLs that affected lint fiber productivity in the same populations, perhaps suggesting pleiotropic effects on both fiber types. However, only Li ₁ mapped within the likelihood interval for 191 previously detected lint fiber QTLs discovered in non-mutant crosses, suggesting that these mutations may occur in genes that played early roles in cotton fiber evolution, and for which new allelic variants are quickly eliminated from improved germplasm. A close positional association between sma-4(h _a ), two leaf and stem-borne trichome mutants (t ₁ , t ₂), and a gene previously implicated in fiber development, sucrose synthase, raises questions about the possibility that these genes may be functionally related. Increasing knowledge of the correspondence of the cotton and Arabidopsis genomes provides several avenues by which genetic dissection of cotton fiber development may be accelerated.     

Hypodermal expression of Caenorhabditis elegans TGF-beta type I receptor SMA-6 is essential for the growth and maintenance of body length.

There are several transforming growth factor-beta (TGF-beta) pathways in the nematode Caenorhabditis elegans. One of these pathways regulates body length and is composed of the ligand DBL-1, serine/threonine protein kinase receptors SMA-6 and DAF-4, and cytoplasmic signaling components SMA-2, SMA-3, and SMA-4. To further examine the molecular mechanisms of body-length regulation in the nematode by the TGF-beta pathway, we examined the regional requirement for the type-I receptor SMA-6. Using a SMA-6::GFP (green fluorescent protein) reporter gene, sma-6 was highly expressed in the hypodermis, unlike the type-II receptor DAF-4, which is reported to be ubiquitously expressed. We then examined the ability of SMA-6 expression in different regions of the C. elegans body to rescue the sma-6 phenotype (small) and found that hypodermal expression of SMA-6 is necessary and sufficient for the growth and maintenance of body length. We also demonstrate that GATA sequences in the sma-6 promoter contribute to the hypodermal expression of sma-6.     

Hair,encoding a single C2H2 zinc‐finger protein,regulates multicellular trichome formation in tomato

Trichomes originate from the epidermal cells of nearly all terrestrial plants, which are specialized unicellular or multicellular structures. Although the molecular mechanism regulating unicellular trichome formation has been extensively characterized, most of the genes essential for multicellular trichome formation remain unknown. In this study, we identified an associated locus on the long arm of chromosome 10 using a genome‐wide association study (GWAS) on type‐I trichomes of 180 diverse Solanum lycopersicum (tomato) accessions. Using map‐based cloning we then cloned the key gene controlling the initiation of this type of trichome, named Hair (H), which encodes a single C2H2 zinc‐finger protein. Transgenic experiments showed that hair‐absent phenotype is caused by the deletion of the entire coding region of H. We identified three alleles of H containing several missense mutations and a nucleotide deletion, which result in amino acid substitutions and a reading frame shift, respectively. In addition, knockdown of H or Woolly (Wo) represses the formation of type‐I trichomes, suggesting that both regulators may function as a heterodimer. Direct protein–protein interaction between them was further detected through pull‐down and yeast two‐hybrid assays. In addition, ectopic expression of H in Nicotiana tabacum (tobacco) and expression of its homologs from Capsicum annuum (pepper) and tobacco in tomato can trigger trichome formation. Taken together, these findings suggest that the H gene may be functionally conserved in multicellular trichome formation in Solanaceae species.     

Genetic interaction between AINTEGUMENTA (ANT) and the ovule identity genes SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2)

AINTEGUMENTA (ANT) promotes initiation and growth of ovule integuments which cell fate is specified by ovule identity factors, such as SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2). To study the genetic interaction between ANT and the ovule identity genes, we have obtained a stk shp1 shp2 ant quadruple mutant. The molecular and morphological characterization of the quadruple mutant and its comparison with the stk shp1 shp2 triple mutant, the shp1 shp2 ant triple mutant and the stk ant double mutant are here presented.     

Interaction between HY1 and H2O2 in auxin-induced lateral root formation in Arabidopsis

Haem oxygenase-1 (HO-1) and hydrogen peroxide (H₂O₂) are two key downstream signals of auxin, a well-known phytohormone regulating plant growth and development. However, the inter-relationship between HO-1 and H₂O₂ in auxin-mediated lateral root (LR) formation is poorly understood. Herein, we revealed that exogenous auxin, 1-naphthylacetic acid (NAA), could simultaneously stimulate Arabidopsis HO-1 (HY1) gene expression and H₂O₂ generation. Subsequently, LR formation was induced. NAA-induced HY1 expression is dependent on H₂O₂. This conclusion was supported by analyzing the removal of H₂O₂ with ascorbic acid (AsA) and dimethylthiourea (DMTU), both of which could block NAA-induced HY1 expression and LR formation. H₂O₂-induced LR formation was inhibited by an HO-1 inhibitor zinc protoporphyrin IX (Znpp) in wild-type and severely impaired in HY1 mutant hy1-100. Simultaneously, HY1 is required for NAA-mediated H₂O₂ generation, since Znpp inhibition of HY1 blocked the NAA-induced H₂O₂ production and LR formation. Genetic data demonstrated that hy1-100 was significantly impaired in H₂O₂ production and LR formation in response to NAA, compared with wild-type plants. The addition of carbon monoxide-releasing molecule-2 (CORM-2), the carbon monoxide (CO) donor, induced H₂O₂ production and LR formation, both of which were decreased by DMTU. Moreover, H₂O₂ and CORM-2 mimicked the NAA responses in the regulation of cell cycle genes expression, all of which were blocked by Znpp or DMTU, respectively, confirming that both H₂O₂ and CO were important in the early LR initiation. In summary, our pharmacological, genetic and molecular evidence demonstrated a close inter-relationship between HY1 and H₂O₂ existing in auxin-induced LR formation in Arabidopsis.     

Salicylic acid antagonism of EDS1‐driven cell death is important for immune and oxidative stress responses in Arabidopsis

Reactive oxygen species (ROS) have emerged as signals in the responses of plants to stress. Arabidopsis Enhanced Disease Susceptibility1 (EDS1) regulates defense and cell death against biotrophic pathogens and controls cell death propagation in response to chloroplast‐derived ROS. Arabidopsis Nudix hydrolase7 (nudt7) mutants are sensitized to photo‐oxidative stress and display EDS1‐dependent enhanced resistance, salicylic acid (SA) accumulation and initiation of cell death. Here we explored the relationship between EDS1, EDS1‐regulated SA and ROS by examining gene expression profiles, photo‐oxidative stress and resistance phenotypes of nudt7 mutants in combination with eds1 and the SA‐biosynthetic mutant, sid2. We establish that EDS1 controls steps downstream of chloroplast‐derived O₂^?? that lead to SA‐assisted H₂O₂ accumulation as part of a mechanism limiting cell death. A combination of EDS1‐regulated SA‐antagonized and SA‐promoted processes is necessary for resistance to host‐adapted pathogens and for a balanced response to photo‐oxidative stress. In contrast to SA, the apoplastic ROS‐producing enzyme NADPH oxidase RbohD promotes initiation of cell death during photo‐oxidative stress. Thus, chloroplastic O₂^?? signals are processed by EDS1 to produce counter‐balancing activities of SA and RbohD in the control of cell death. Our data strengthen the idea that EDS1 responds to the status of O₂^?? or O₂^??‐generated molecules to coordinate cell death and defense outputs. This activity may enable the plant to respond flexibly to different biotic and abiotic stresses in the environment.     

Overexpression of Peroxisomal Malate Dehydrogenase MDH3 Gene Enhances Cell Death on H 2O 2 Stress in the ald5 Mutant of Saccharomyces cerevisiae

Mitochondrial aldehyde dehydrogenase ALD5 of Saccharomyces cerevisiae is involved in the biosynthesis of mitochondrial electron transport chain, and the ald5 mutant is incompetent for respiration. With use of the mutant, we examined the detoxication of H₂O₂ generation by fatty acid -oxidation in peroxisome. The ald5 mutant (AKD321), as well as the 746 ⁰ mutant, was more resistant to H₂O₂ stress than the wild type. However, overexpression of the MDH3 gene that was involved in the reoxidation of NADH during fatty acid -oxidation caused a decrease in cell viability of AKD321 to H₂O₂ stress, while the 746 ⁰ mutant had no such effect. Intracellular H₂O₂ concentration increased approximately fourfold in MDH3 overexpressing ald5 strain (MD3-AKD321), compared with AKD321. The peroxisomal catalase activity of MD3-AKD321 decreased by 83% to that of AKD321. And also, the overexpression of MDH3 had only a weak effect in MDH3 overexpressing 746 ⁰ strain, decreasing by 14% to that of 746 ⁰ mutant. The increased palmitoyl CoA oxidation by overexpression of MDH3 gene was the same in both strains. Under conditions of MDH3 overexpression, peroxisomal catalase (CTA1) appears to be a limiting factor to oxidative stress. These observations point to an important, as yet unidentified, role of mitochondrial aldehyde dehydrogenase (ALD5) to endogeneous oxidative stress in peroxisome.Received: 23 September 2002 / Accepted: 24 October 2002     

SMA-1 spectrin has essential roles in epithelial cell sheet morphogenesis in C. elegans

During Caenorhabditis elegans development, the embryo acquires its vermiform shape due to changes in the shape of epithelial cells, a process that requires an apically localized actin cytoskeleton. We show that SMA-1, an ortholog of beta(H)-spectrin required for normal morphogenesis, localizes to the apical membrane of epithelial cells when these cells are rapidly elongating. In spc-1 alpha-spectrin mutants, SMA-1 localizes to the apical membrane but its organization is altered, consistent with the hypothesis these proteins act together to form an apically localized spectrin-based membrane skeleton (SBMS). SMA-1 is required to maintain the association between actin and the apical membrane; sma-1 mutant embryos fail to elongate because actin, which provides the driving force for cell shape change, dissociates from the apical membrane skeleton during morphogenesis. Analysis of sma-1 expression constructs and mutant strains indicates SMA-1 maintains the association between actin and the apical membrane via interactions at its N-terminus and this activity is independent of alpha-spectrin. SMA-1 also preserves dynamic changes in the organization of the apical membrane skeleton. Taken together, our results show the SMA-1 SBMS plays a dynamic role in converting changes in actin organization into changes in epithelial cell shape during C. elegans embryogenesis.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

The nuclear transporter SAD2 plays a role in calcium‐ and H2O2‐mediated cell death in Arabidopsis

In response to pathogens, plant cells exhibit a rapid increase in the intracellular calcium concentration and a burst of reactive oxygen species (ROS). The cytosolic increase in Ca²⁺ and the accumulation of ROS are critical for inducing programmed cell death (PCD), but the molecular mechanism is not fully understood. We screened an Arabidopsis mutant, sad2‐5, which harbours a T‐DNA insertion in the 18^th exon of the importin beta‐like gene, SAD2. The H₂O₂‐induced increase in the [Ca²⁺]_cyt of the sad2‐5 mutant was greater than that of the wild type, and the sad2‐5 mutant showed clear cell death phenotypes and abnormal H₂O₂ accumulation under fumonisin‐B1 (FB1) treatment. CaCl₂ could enhance the FB1‐induced cell death of the sad2‐5 mutant, whereas lanthanum chloride (LaCl₃), a broad‐spectrum calcium channel blocker, could restore the FB1‐induced PCD phenotype of sad2‐5. The sad2‐5 fbr11‐1 double mutant exhibited the same FB1‐insensitive phenotype as fbr11‐1, which plays a critical role in novo sphingolipid synthesis, indicating that SAD2 works downstream of FBR11. These results suggest the important role of nuclear transporters in calcium‐ and ROS‐mediated PCD response as well as provide an important theoretical basis for further analysis of the molecular mechanism of SAD2 function in PCD and for improvement of the resistance of crops to adverse environments.     

In planta measurements of oxidative bursts elicited by avirulent and virulent bacterial pathogens suggests that H2O2 is insufficient to elicit cell death in tobacco

A rapid and localized programmed cell death – the hypersensitive response (HR) – is a widely utilized plant resistance mechanism against pathogens. Studies have implicated H₂O₂ generation as a key elicitory mechanism in the HR. The causal relationship between the kinetics of the in planta oxidative burst, the HR and certain defence gene expression was examined. H₂O₂ generation following challenge with avirulent strains of Pseudomonas syringae pv. (P. s. pv.) syringae occurred in two phases. The effects of ROS generation were investigated using the H₂O₂-responsive transgene AoPR10-GUS, the dually responsive (H₂O₂ and salicylic acid) PR1a-GUS as well as measures of cell death. Co-application of catalase with P. s. pv. syringae into tobacco leaf panels suppressed AoPR10- and PR1a-GUS expression and cell death. Conversely, varying H₂O₂ generation with glucose: glucose oxidase influenced both defence gene expression and cell death. AoPR10-GUS proved to be primarily responsive to apoplastic not intracellular oxidative stress, suggesting that the apoplasm was a distinctive source of oxidative signals. A biphasic oxidative burst was also observed with virulent P. s. pv. tabaci, which, although delayed compared to that observed during HR, persisted at equivalent levels for a longer period. Taking all these data together we suggest that either (1) additional factors to the apoplastic oxidative burst are required to explain the rapid kinetics of defence signalling and cell death associated with the HR or (2) P. s. pv. tabaci successfully suppresses the effects of H₂O₂ generation by an unknown mechanism.