菠萝蜜白化突变体叶片表皮形态研究

为探究木本植物白化突变体叶片表皮形态的变化,在扫描电镜下观测了菠萝蜜（Artocarpus heterophyllus）白化突变体（AAS）和正常（CK）幼苗叶片的表皮细胞和气孔器,对MAP65家族蛋白构建了进化树,并分析了MAP65基因的表达模式。结果表明,AAS表皮细胞和气孔器的大小、形态均发生较大变化。与CK相比,AAS表皮细胞的周长、面积较小,密度较大,凸出数量和长度均减少,气孔器较小且大小不一。下表皮小细胞和异常气孔器的数量在AAS中大幅增加。MAP65家族成员大部分基因在AAS中下调表达。因此,推测菠萝蜜白化突变体的发生可能与MAP65基因表达有关。     

Sterols are required for cell‐fate commitment and maintenance of the stomatal lineage in Arabidopsis

Asymmetric cell division is important for regulating cell proliferation and fate determination during stomatal development in plants. Although genes that control asymmetric division and cell differentiation in stomatal development have been reported, regulators controlling the process from asymmetric division to cell differentiation remain poorly understood. Here, we report a weak allele (fk–J3158) of the Arabidopsis sterol C–14 reductase gene FACKEL (FK) that shows clusters of small cells and stomata in leaf epidermis, a common phenomenon that is often seen in mutants defective in stomatal asymmetric division. Interestingly, the physical asymmetry of these divisions appeared to be intact in fk mutants, but the cell‐fate asymmetry was greatly disturbed, suggesting that the FK pathway links these two crucial events in the process of asymmetric division. Sterol profile analysis revealed that the fk–J3158 mutation blocked downstream sterol production. Further investigation indicated that cyclopropylsterol isomerase1 (cpi1), sterol 14α–demethylase (cyp51A2) and hydra1 (hyd1) mutants, corresponding to enzymes in the same branch of the sterol biosynthetic pathway, displayed defective stomatal development phenotypes, similar to those observed for fk. Fenpropimorph, an inhibitor of the FK sterol C–14 reductase in Arabidopsis, also caused these abnormal small‐cell and stomata phenotypes in wild‐type leaves. Genetic experiments demonstrated that sterol biosynthesis is required for correct stomatal patterning, probably through an additional signaling pathway that has yet to be defined. Detailed analyses of time‐lapse cell division patterns, stomatal precursor cell division markers and DNA ploidy suggest that sterols are required to properly restrict cell proliferation, asymmetric fate specification, cell‐fate commitment and maintenance in the stomatal lineage cells. These events occur after physical asymmetric division of stomatal precursor cells.     

Direct measurement of stomatal cell production in barley

Summary A simple method which measures stomatal cell production directly in the first leaf of barley is described. The method was tested with three barley genotypes, two of which, cer-g ¹¹ and cer-g ¹⁰²⁶, are mutants with an abnormal stomatal pattern. Cer-g ¹¹ had a slower rate of stomatal cell formation, as compared with the other two genotypes.     

MPK9 and MPK12 function in SA-induced stomatal closure in Arabidopsis thaliana

Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.     

Developmental defects and seedling lethality in apyrase <Emphasis Type="Italic">AtAPY1</Emphasis> and <Emphasis Type="Italic">AtAPY2</Emphasis> double knockout mutants

Previously it was shown that the Arabidopsis apyrase genes AtAPY1 and AtAPY2 are crucial for male fertility because mutant pollen (apy1-1; apy2-1) with T-DNA insertions in both genes could not germinate (Steinebrunner et al. (2003) Plant Physiol. 131: 1638–1647). In this study, pollen germination was restored and apyrase T-DNA double knockouts (DKO) apy1-1/apy1-1; apy2-1/apy2-1 were generated by complementation with AtAPY2 under the control of a pollen-specific promoter. The DKO phenotype displayed developmental defects including the lack of functional root and shoot meristems. In cotyledons, morphogenetic and patterning abnormalities were apparent, e.g., unlobed pavement cells and stomatal clusters. Another set of lines was created which carried either AtAPY1 or AtAPY2 under a dexamethasone-(DEX)-inducible promoter as an additional transgene to the pollen-specific gene construct. Application of DEX did not reverse the DKO phenotype to wild-type, but some inducible lines exhibited less severe defects even in the absence of the inducer, probably due to some background expression. However, even these DKO mutants were seedling-lethal and shared other defects regarding cell division, cell expansion and stomatal patterning. Taken together, the defects in the DKO mutants demonstrate that AtAPY1 and AtAPY2 are essential for normal plant development.     

Late embryo-defective mutants of Arabidopsis

The embryo-defective (emb) mutants of Arabidopsis constitute a large and diverse group of mutants disrupted in a broad range of embryonic processes, including morphogonesis, cell differentiation, and maturation programs. This report describes a subset of these mutants, the late embryo defectives, which develop beyond the globular stage of embryogenesis but fail to complete normal morphogenesis. A representative sample of 12 late mutants was chosen for this study, patterns of morphogenesis were characterized, the germination potential of mutant seeds was investigated, and additional mutant alleles within the collection were identified. Morphological defects in mutant embryos became apparent during the heart stage of development, when embryos normally begin the rapid cell division and expansion required for the completion of morphogenesis. Despite their morphological abnormalities, mutant embryos often germinated from dry seed, demonstrating that genetic programs required for the establishment of desiccation tolerance remained intact. Mutant seedlings displayed a wide range of developmental abnormalities, including altered morphology, lack of pigmentation, dwarfism, and disorganized vegetative growth. One late mutant was found to be allelic to an early embryo defective that arrests at the globular stage. These results suggest that a number of late EMB genes encode basic cellular and metabolic functions needed for cell division, enlargement, and embryonic growth. The rapid growth and metabolic changes that occur at the heart stage may present a barrier to normal development in the late mutants, resulting in altered embryo morphology and other developmental defects. It is proposed that many Arabidopsis mutants with abnormal embryo and seedling morphology are not defective in the regulation of pattern formation or morphogenesis, but rather in fundamental physiological and cellular processes required for the completion of normal growth and development. © 1995 Wiley-Liss, Inc.     

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin‐related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin‐D‐induced depolymerization or phalloidin‐induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild‐type (WT) Arabidopsis plants. Light‐induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.     

Stomatal function,density and pattern,and CO2 assimilation in Arabidopsis thaliana tmm1 and sdd1‐1 mutants

• Stomata modulate the exchange of water and CO₂ between plant and atmosphere. Although stomatal density is known to affect CO₂ diffusion into the leaf and thus photosynthetic rate, the effect of stomatal density and patterning on CO₂ assimilation is not fully understood.
• We used wild types Col‐0 and C24 and stomatal mutants sdd1‐1 and tmm1 of Arabidopsis thaliana, differing in stomatal density and pattern, to study the effects of these variations on both stomatal and mesophyll conductance and CO₂ assimilation rate. Anatomical parameters of stomata, leaf temperature and carbon isotope discrimination were also assessed.
• Our results indicate that increased stomatal density enhanced stomatal conductance in sdd1‐1 plants, with no effect on photosynthesis, due to both unchanged photosynthetic capacity and decreased mesophyll conductance. Clustering (abnormal patterning formed by clusters of two or more stomata) and a highly unequal distribution of stomata between the adaxial and abaxial leaf sides in tmm1 mutants also had no effect on photosynthesis.
• Except at very high stomatal densities, stomatal conductance and water loss were proportional to stomatal density. Stomatal formation in clusters reduced stomatal dynamics and their operational range as well as the efficiency of CO₂ transport.

     

Developmental mutants showing abnormal organ differentiation in rice embryos

Summary Zygotes of rice (Oryza sativa L. cv Taichung 65) were treated with 1.0 mM solution of the chemical mutagen N-methyl-N-nitrosourea. Out of 1420 M₂ lines, 28 single-locus recessive mutants on embryogenesis were identified. Among them, we analyzed 11 mutants in the present study, which differentiated the shoot (plumule) and/or root (radicle) with abnormality. Of the 11 mutants, two showed no shoot differentiation with normal root. On the other hand, we could not detect any mutant which exhibited a normal shoot without a root. This suggests that shoot and root are genetically controlled by different loci and that the alleles associated with shoot formation mutate more frequently than do those of the root. Five mutants showed aberrant morphology of shoot when both the shoot and root developed. One of them, odm 5 (organ differententiation mutant 5) was germinable, but produced many fine and twisted leaves. This mutant was, however, lethal at the early post-germination stage under the usual cultural conditions. In another mutant (odm 4), shoot differentiation seemed to be initiated at an arbitrary position, resulting in a very abnormal morphology of the shoot when the position fronted the endosperm. The other two mutants showed abnormal morphology of both the shoot and root. One (odm 11) of the remaining two mutants showed a wide variation of abnormalities including no organ differentiation, either shoot or root differentiation and the development of both shoot and root with abnormalities. The last one (odm 16) was unique. It had an embryo with normal shoot and root but the embryo size was only one-third to one-half of normal embryos in length. Of course, the shoot and root are also small but viable. Therefore, odm 16 is considered to be a mutant in the size regulation of the embryo. Although an allelism test has not yet been done, most of these mutants are probably non-allelic, as the phenotypic abnormality differs largely with each one. In rice, the shoot and root highly differentiate in contrast to dicotyledonous embryo. Accordingly, these developmental mutants are very useful materials for investigating the regulatory mechanism of gene expression in organ differentiation.     

CYTOLOGICAL STUDIES OF NONTRUE–BREEDING MUTANTS IN SORGHUM OBTAINED AFTER COLCHICINE TREATMENT

Sanders , Mary E., and Clifford J. Franzke . (South Dakota State Coll.. Brookings.) Cytological studies of nontrue-breeding mutants in sorghum obtained after colchicine treatment. Amer. Jour. Bot. 49(9): 990–996. Illus. 1962.—Although pollen mother cells of nontrue-breeding mutant plants obtained after colchicine treatment of sorghum seedlings, line ‘Experimental 3,’ showed normal chromosome behavior (10 bivalents) at phases of meiosis I, some abnormalities were found at corresponding stages in first- and second-generation self-progeny plants of some of them. The most frequent chromosome irregularities were an increase over ‘Experimental 3’ in number of cells containing univalents, and mixoploid tissues with tetraploid and diploid cells. The higher polyploid groups (6n, 8n, 10n) also present in 2 plants might be related to their male-sterile condition rather than being an indication of the chromosome complement. Abnormalities in progenies suggest that some of the mutants might have been chimeras in which abnormalities were missed and raise the question whether chromosome changes are involved in the formation of the mutants in spite of the preponderance of normal diploid cells with 10 bivalents during prophase and metaphase of meiosis I. This could occur if sorghum contains a genetic mechanism which promotes bivalent rather than multivalent pairing. That such might be a possibility is indicated by the large numbers of bivalents and small numbers of multivalents found in polyploid cells or groups.     

Heterotrimeric G protein mediates ethylene‐induced stomatal closure via hydrogen peroxide synthesis in Arabidopsis

Heterotrimeric G proteins function as key players in hydrogen peroxide (H₂O₂) production in plant cells, but whether G proteins mediate ethylene‐induced H₂O₂ production and stomatal closure are not clear. Here, evidences are provided to show the Gα subunit GPA1 as a missing link between ethylene and H₂O₂ in guard cell ethylene signalling. In wild‐type leaves, ethylene‐triggered H₂O₂ synthesis and stomatal closure were dependent on activation of Gα. GPA1 mutants showed the defect of ethylene‐induced H₂O₂ production and stomatal closure, whereas wGα and cGα overexpression lines showed faster stomatal closure and H₂O₂ production in response to ethylene. Ethylene‐triggered H₂O₂ generation and stomatal closure were impaired in RAN1, ETR1, ERS1 and EIN4 mutants but not impaired in ETR2 and ERS2 mutants. Gα activator and H₂O₂ rescued the defect of RAN1 and EIN4 mutants or etr1‐3 in ethylene‐induced H₂O₂ production and stomatal closure, but only rescued the defect of ERS1 mutants or etr1‐1 and etr1‐9 in ethylene‐induced H₂O₂ production. Stomata of CTR1 mutants showed constitutive H₂O₂ production and stomatal closure, but which could be abolished by Gα inhibitor. Stomata of EIN2, EIN3 and ARR2 mutants did not close in responses to ethylene, Gα activator or H₂O₂, but do generate H₂O₂ following challenge of ethylene or Gα activator. The data indicate that Gα mediates ethylene‐induced stomatal closure via H₂O₂ production, and acts downstream of RAN1, ETR1, ERS1, EIN4 and CTR1 and upstream of EIN2, EIN3 and ARR2. The data also show that ETR1 and ERS1 mediate both ethylene and H₂O₂ signalling in guard cells.     

Neuronal development in the Drosophila compound eye: Photoreceptor cells R1, R6, and R7 fail to differentiate in the retina aberrant in pattern (rap) mutant

The compound eye of Drosophila is a reiterated pattern of 800 unit eyes known as ommatidia. In each ommatidium there are eight photoreceptor neurons (R1–R8) and an invariant number of accessory cells organized in a precise manner. In the developing eye, specification of cell fates is triggered by sequential inductive events mediated by cell-cell interactions. The R8 photoreceptor neuron is the first cell to differentiate and is thought to play a central role in the recruitment of the remaining photoreceptor cells. Our previous work demonstrated that mutations in the retina aberrant in pattern (rap) locus lead to abnormal pattern formation in the compound eye. Genetic mosaic experiments demonstrated that for normal retinal patterning to occur, rap gene function is required only in the photoreceptor cell R8. In this study we analyzed the R cell composition of developing as well as the adult eyes of rap mutants employing a variety of R cell specific markers. We show that in rap mutants, although some of the R8-specific markers show normal expression patterns, other aspects of the R8 cell differentiation are abnormal. In addition, the cells R1, R6, and R7 fail to differentiate properly in rap mutants. These results suggest that the rap gene encodes an R8-specific function that plays a role in the determination of the photoreceptor cells R1, R6, and R7. © 1996 John Wiley & Sons, Inc.     

Deficient Glutathione in Guard Cells Facilitates Abscisic Acid-Induced Stomatal Closure but Does Not Affect Light-Induced Stomatal Opening

We investigated the role of glutathione (GSH) in stomatal movements using a GSH deficient mutant, chlorinal-1 (ch1-1). Guard cells of ch1-1 mutants accumulated less GSH than wild types did. Light induced stomatal opening in ch1-1 and wild-type plants. Abscisic acid (ABA) induced stomatal closure in ch1-1 mutants more than wild types without enhanced reactive oxygen species (ROS) production. Therefore, GSH functioned downstream of ROS production in the ABA signaling cascade.     

Jasmonate‐mediated stomatal closure under elevated CO2 revealed by time‐resolved metabolomics

Foliar stomatal movements are critical for regulating plant water loss and gas exchange. Elevated carbon dioxide (CO₂) levels are known to induce stomatal closure. However, the current knowledge on CO₂ signal transduction in stomatal guard cells is limited. Here we report metabolomic responses of Brassica napus guard cells to elevated CO₂ using three hyphenated metabolomics platforms: gas chromatography‐mass spectrometry (MS); liquid chromatography (LC)‐multiple reaction monitoring‐MS; and ultra‐high‐performance LC‐quadrupole time‐of‐flight‐MS. A total of 358 metabolites from guard cells were quantified in a time‐course response to elevated CO₂ level. Most metabolites increased under elevated CO₂, showing the most significant differences at 10 min. In addition, reactive oxygen species production increased and stomatal aperture decreased with time. Major alterations in flavonoid, organic acid, sugar, fatty acid, phenylpropanoid and amino acid metabolic pathways indicated changes in both primary and specialized metabolic pathways in guard cells. Most interestingly, the jasmonic acid (JA) biosynthesis pathway was significantly altered in the course of elevated CO₂ treatment. Together with results obtained from JA biosynthesis and signaling mutants as well as CO₂ signaling mutants, we discovered that CO₂‐induced stomatal closure is mediated by JA signaling.     

PHO1 expression in guard cells mediates the stomatal response to abscisic acid in Arabidopsis

Stomatal opening and closing are driven by ion fluxes that cause changes in guard cell turgor and volume. This process is, in turn, regulated by environmental and hormonal signals, including light and the phytohormone abscisic acid (ABA). Here, we present genetic evidence that expression of PHO1 in guard cells of Arabidopsis thaliana is required for full stomatal responses to ABA. PHO1 is involved in the export of phosphate into the root xylem vessels and, as a result, the pho1 mutant is characterized by low shoot phosphate levels. In leaves, PHO1 was found expressed in guard cells and up‐regulated following treatment with ABA. The pho1 mutant was unaffected in production of reactive oxygen species following ABA treatment, and in stomatal movements in response to light cues, high extracellular calcium, auxin, and fusicoccin. However, stomatal movements in response to ABA treatment were severely impaired, both in terms of induction of closure and inhibition of opening. Micro‐grafting a pho1 shoot scion onto wild‐type rootstock resulted in plants with normal shoot growth and phosphate content, but failed to restore normal stomatal response to ABA treatment. PHO1 knockdown using RNA interference specifically in guard cells of wild‐type plants caused a reduced stomatal response to ABA. In agreement, specific expression of PHO1 in guard cells of pho1 plants complemented the mutant guard cell phenotype and re‐established ABA sensitivity, although full functional complementation was dependent on shoot phosphate sufficiency. Together, these data reveal an important role for phosphate and the action of PHO1 in the stomatal response to ABA.     

Lethal(1)aberrant immune response mutations leading to melanotic tumor formation in Drosophila melanogaster

Using P element-mediated mutagenesis we have isolated 20 X-linked lethal mutations, representing at least 14 complementation groups, which exhibit melanotic tumor phenotypes. We present the systematic analysis of this interesting group of lethal mutations that were selected for their visible melanotic or immune response. The lethal and melanotic tumor phenotypes of each lethal(1) aberrant immune response (air) mutation are pleiotropic effects of single genetic lesions. Lethality occurs throughout the larval and early pupal periods of development and larval development is extended in some air mutants. The air mutant lethal syndromes include abnormalities associated with the brain, haematopoietic organs, gut, salivary glands, ring glands, and imaginal discs. Additional characterization of the melanotic tumor mutations Tum^l and tu(1)Sz^ts have indicated that the melanotic tumor phenotype is similar to that observed in the air mutants. These studies have led to the proposal that two distinct classes of melanotic tumor mutations exist. Class 1 includes mutants in which melanotic tumors result from “autoimmune responses” or the response of an apparently normal immune system to the presence of abnormal target tissues. The Class 2 mutants display obvious defects in the haematopoietic organs or haemocytes, manifested as overgrowth, and the resulting aberrant immune system behavior may contribute to melanotic tumor formation.     

Two guard cell‐preferential MAPKs,MPK9 and MPK12, regulate YEL signalling in Arabidopsis guard cells

We report that two mitogen‐activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate abscisic acid (ABA)‐induced stomatal closure in Arabidopsis thaliana. Yeast elicitor (YEL) induced stomatal closure accompanied by intracellular reactive oxygen species (ROS) accumulation and cytosolic free calcium concentration ([Ca²⁺]_cyt) oscillation. In this study, we examined whether these two MAP kinases are involved in YEL‐induced stomatal closure using MAPKK inhibitors, PD98059 and U0126, and MAPK mutants, mpk9, mpk12 and mpk9 mpk12. Both PD98059 and U0126 inhibited YEL‐induced stomatal closure. YEL induced stomatal closure in the mpk9 and mpk12 mutants but not in the mpk9 mpk12 mutant, suggesting that a MAPK cascade involving MPK9 and MPK12 functions in guard cell YEL signalling. However, YEL induced extracellular ROS production, intracellular ROS accumulation and cytosolic alkalisation in the mpk9, mpk12 and mpk9 mpk12 mutants. YEL induced [Ca²⁺]_cyt oscillations in both wild type and mpk9 mpk12 mutant. These results suggest that MPK9 and MPK12 function redundantly downstream of extracellular ROS production, intracellular ROS accumulation, cytosolic alkalisation and [Ca²⁺]_cyt oscillation in YEL‐induced stomatal closure in Arabidopsis guard cells and are shared with ABA signalling.     

Abnormal stomatal behavior and root resistance,and hormonal imbalance in three wilty mutants of tomato

Three wilty mutants of tomato, flacca, sitiens, and notabilis, were compared with two control normal cultivars, Rheinlands Ruhm and Lukullus, for concentration of abscisic acid and root resistance to water flow. In addition, the three mutants treated with abscisic acid during development were compared with control mutant plants for stomatal opening and root resistance. The hormonal concentration was estimated by coleoptile assay and gas chromatography. Stomatal opening was estimated by measuring rate of transpiration and by examining leaf imprints. Root resistance was estimated by measuring the amount of exudate from roots of decapitated plants and the difference between the osmotic pressure of the exudate and the root medium. A lower level of abscisic acid was found in all three mutants as compared with the control normal plants. In addition, root resistance to water flow was higher in the three mutants than in the control normal types. All three mutants completely reverted to normal phenotypically, including stomatal and root resistances, when treated with abscisic acid. It has been suggested that the first hormonal change in the mutants is that of abscisic acid, and from it proceed the other changes.This work was supported in part by a research grant from the Ford Foundation (Ford-6, A-VII).