Synergistic interaction of the two paralogous Arabidopsis genes LRX1 and LRX2 in cell wall formation during root hair development

LRR-extensins (LRX) form a family of structural cell wall proteins containing a receptor-like domain. The functional analysis of Arabidopsis LRX1 has shown that it is involved in cell morphogenesis of root hairs. In this work, we have studied LRX2, a paralog of LRX1. LRX2 expression is mainly found in roots and is responsive to factors promoting or repressing root hair formation. The function of LRX1 and LRX2 was tested by the expression of a truncated LRX2 and different LRX1/LRX2 chimaeric proteins. Using complementation of the lrx1 phenotype as the parameter for protein function, our experiments indicate that LRX1 and LRX2 are functionally similar but show differences in their activity. Genetic analysis revealed that single lrx2 mutants do not show any defect in root hair morphogenesis, but synergistically interact with the lrx1 mutation. lrx1/lrx2 double mutants have a significantly enhanced lrx1 phenotype, resulting in frequent rupture of the root hairs soon after their initiation. Analysis of the root hair cell wall ultrastructure by transmission electron microscopy (TEM) revealed the formation of osmophilic aggregates within the wall, as well as local disintegration of the wall structure in the double mutant, but not in wild-type plants. Our results indicate that LRX1 and LRX2 have overlapping functions in root hair formation, and that they likely regulate cell morphogenesis by promoting proper development of the cell wall.     

Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product.

We characterized the distribution of AGAMOUS (AG) RNA during early flower development in Arabidopsis. Mutations in this homeotic gene cause the transformation of stamens to petals in floral whorl 3 and of carpels to another ag flower in floral whorl 4. We found that AG RNA is present in the stamen and carpel primordia but is undetectable in sepal and petal primordia throughout early wild-type flower development, consistent with the mutant phenotype. We also analyzed the distribution of AG RNA in apetela2 (ap2) mutant flowers. AP2 is a floral homeotic gene that is necessary for the normal development of sepals and petals in floral whorls 1 and 2. In ap2 mutant flowers, AG RNA is present in the organ primordia of all floral whorls. These observations show that the expression patterns of the Arabidopsis floral homeotic genes are in part established by regulatory interactions between these genes.     

Maturation of arabidopsis seeds is dependent on glutathione biosynthesis within the embryo

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.     

ROXY1, a member of the plant glutaredoxin family, is required for petal development in Arabidopsis thaliana

We isolated three alleles of an Arabidopsis thaliana gene named ROXY1, which initiates a reduced number of petal primordia and exhibits abnormalities during further petal development. The defects are restricted to the second whorl of the flower and independent of organ identity. ROXY1 belongs to a subgroup of glutaredoxins that are specific for higher plants and we present data on the first characterization of a mutant from this large Arabidopsis gene family for which information is scarce. ROXY1 is predominantly expressed in tissues that give rise to new flower primordia, including petal precursor cells and petal primordia. Occasionally, filamentous organs with stigmatic structures are formed in the second whorl of the roxy1 mutant, indicative for an ectopic function of the class C gene AGAMOUS (AG). The function of ROXY1 in the negative regulation of AG is corroborated by premature and ectopic AG expression in roxy1-3 ap1-10 double mutants, as well as by enhanced first whorl carpeloidy in double mutants of roxy1 with repressors of AG, such as ap2 or lug. Glutaredoxins are oxidoreductases that oxidize or reduce conserved cysteine-containing motifs. Mutagenesis of conserved cysteines within the ROXY1 protein demonstrates the importance of cysteine 49 for its function. Our data demonstrate that, unexpectedly, a plant glutaredoxin is involved in flower development, probably by mediating post-translational modifications of target proteins required for normal petal organ initiation and morphogenesis.     

SCD1 is required for cytokinesis and polarized cell expansion in Arabidopsis thaliana [corrected

In the leaf epidermis, guard mother cells undergo a stereotyped symmetric division to form the guard cells of stomata. We have identified a temperature-sensitive Arabidopsis mutant, stomatal cytokinesis-defective 1-1 (scd1-1), which affects this specialized division. At the non-permissive temperature, 22 degrees C, defective scd1-1 guard cells are binucleate, and the formation of their ventral cell walls is incomplete. Cytokinesis was also disrupted in other types of epidermal cells such as pavement cells. Further phenotypic analysis of scd1-1 indicated a role for SCD1 in seedling growth, root elongation and flower morphogenesis. More severe scd1 T-DNA insertion alleles (scd1-2 and scd1-3) markedly affect polar cell expansion, most notably in trichomes and root hairs. SCD1 is a unique gene in Arabidopsis that encodes a protein related to animal proteins that regulate intracellular protein transport and/or mitogen-activated protein kinase signaling pathways. Consistent with a role for SCD1 in membrane trafficking, secretory vesicles were found to accumulate in cytokinesis-defective scd1 cells. In addition the scd1 mutant phenotype was enhanced by low doses of inhibitors of cell plate consolidation and vesicle secretion. We propose that SCD1 functions in polarized vesicle trafficking during plant cytokinesis and cell expansion.     

Brca2 is involved in meiosis in Arabidopsis thaliana as suggested by its interaction with Dmc1

Two BRCA2-like sequences are present in the Arabidopsis genome. Both genes are expressed in flower buds and encode nearly identical proteins, which contain four BRC motifs. In a yeast two-hybrid assay, the Arabidopsis Brca2 proteins interact with Rad51 and Dmc1. RNAi constructs aimed at silencing the BRCA2 genes at meiosis triggered a reproducible sterility phenotype, which was associated with dramatic meiosis alterations. We obtained the same phenotype upon introduction of RNAi constructs aimed at silencing the RAD51 gene at meiosis in dmc1 mutant plants. The meiotic figures we observed strongly suggest that homologous recombination is highly disturbed in these meiotic cells, leaving aberrant recombination events to repair the meiotic double-strand breaks. The 'brca2' meiotic phenotype was eliminated in spo11 mutant plants. Our experiments point to an essential role of Brca2 at meiosis in Arabidopsis. We also propose a role for Rad51 in the dmc1 context.     

LEAFY controls floral meristem identity in Arabidopsis.

The first step in flower development is the generation of a floral meristem by the inflorescence meristem. We have analyzed how this process is affected by mutant alleles of the Arabidopsis gene LEAFY. We show that LEAFY interacts with another floral control gene, APETALA1, to promote the transition from inflorescence to floral meristem. We have cloned the LEAFY gene, and, consistent with the mutant phenotype, we find that LEAFY RNA is expressed strongly in young flower primordia. LEAFY expression procedes expression of the homeotic genes AGAMOUS and APETALA3, which specify organ identify within the flower. Furthermore, we demonstrate that LEAFY is the Arabidopsis homolog of the FLORICAULA gene, which controls floral meristem identity in the distantly related species Antirrhinum majus.     

The Delayed Terminal Flower Phenotype Is Caused by a Conditional Mutation in the CENTRORADIALIS Gene of Snapdragon

The snapdragon (Antirrhinum majus) centroradialis mutant (cen) is characterized by the development of a terminal flower, thereby replacing the normally open inflorescence by a closed inflorescence. In contrast to its Arabidopsis counterpart, terminal flower1, the cen-null mutant displays an almost constant number of lateral flowers below the terminal flower. Some partial revertants of an X-radiation-induced cen mutant showed a delayed formation of the terminal flower, resulting in a variable number of lateral flowers. The number of lateral flowers formed was shown to be environmentally controlled, with the fewer flowers formed under the stronger flower-inducing conditions. Plants displaying this "Delayed terminal flower" phenotype were found to be heterozygous for a mutant allele carrying a transposon in the coding region and an allele from which the transposon excised, leaving behind a 3-bp duplication as footprint. As a consequence, an iso-leucine is inserted between Asp148 and Gly149 in the CENTRORADIALIS protein. It is proposed that this mutation results in a low level of functional CEN activity, generating a phenotype that is more similar to the Arabidopsis Terminal flower phenotype.     

EMF genes regulate Arabidopsis inflorescence development.

Mutations in EMBRYONIC FLOWER (EMF) genes EMF1 and EMF2 abolish rosette development, and the mutants produce either a much reduced inflorescence or a transformed flower. These mutant characteristics suggest a repressive effect of EMF activities on reproductive development. To investigate the role of EMF genes in regulating reproductive development, we studied the relationship between EMF genes and the genes regulating inflorescence and flower development. We found that APETALA1 and AGAMOUS promoters were activated in germinating emf seedlings, suggesting that these genes may normally be suppressed in wild-type seedlings in which EMF activities are high. The phenotype of double mutants combining emf1-2 and apetala1, apetala2, leafy1, apetala1 cauliflower, and terminal flower1 showed that emf1-2 is epistatic in all cases, suggesting that EMF genes act downstream from these genes in mediating the inflorescence-to-flower transition. Constitutive expression of LEAFY in weak emf1, but not emf2, mutants increased the severity of the emf phenotype, indicating an inhibition of EMF activity by LEAFY, as was deduced from double mutant analysis. These results suggest that a mechanism involving a reciprocal negative regulation between the EMF genes and the floral genes regulates Arabidopsis inflorescence development.     

Key divisions in the early Arabidopsis embryo require POL and PLL1 phosphatases to establish the root stem cell organizer and vascular axis

Arabidopsis development proceeds from three stem cell populations located at the shoot, flower, and root meristems. The relationship between the highly related shoot and flower stem cells and the very divergent root stem cells has been unclear. We show that the related phosphatases POL and PLL1 are required for all three stem cell populations. pol pll1 mutant embryos lack key asymmetric divisions that give rise to the root stem cell organizer and the central vascular axis. Instead, these cells divide in a superficially symmetric fashion in pol pll1 embryos, leading to a loss of embryonic and postembryonic root stem cells and vascular specification. We present data that show that POL/PLL1 drive root stem cell specification by promoting expression of the WUS homolog WOX5. We propose that POL and PLL1 are required for the proper divisions of shoot, flower, and root stem cell organizers, WUS/WOX5 gene expression, and stem cell maintenance.     

拟南芥F-box基因At5g22700的功能初步分析

F-box蛋白作为SCF（Skpl,Cullin and anF-boxprotein）复合体的成员,参与调节植物的生长发育过程。At5g22700为功能未知的F-box基因家族成员。本研究通过酵母双杂交分析At5g22700蛋白与ASK（Arabidop-sis-SKP1-1ike）家族蛋白的相互作用,发现At5g22700蛋白的F-box结构域与ASK4蛋白相互作用。实时定量PCR分析该基因在不同组织器官中的表达,发现该基因在根和花中的表达量最高,说明At5g2700可能在根和花的发育中具有重要作用。以At5g22700基因的T—DNA插入突变体和过量表达转基因株系为材料,分析不同光照条件下幼苗的表型,发现蓝光下At5g22700过量表达转基因幼苗的主根比野生型长。这些研究结果表明,At5g22700在植物体内可能形成SCF复合体,并在植物幼苗主根伸长生长中起促进作用。     

Requirement of KNAT1/BP for the Development of Abscission Zones in Arabidopsis thaliana

The KNAT1 gene is a member of the Class I KNOXhomeobox gene family and is thought to play an important role in meristem development and leaf morphogenesis. Recent studies have demonstrated that KNAT1/BP regulates the architecture of the inflorescence by affecting pedicle development in Arabidopsis thaliana. Herein, we report the characterization of an Arabidopsis T-DNA insertion mutant that shares considerable phenotypic similarity to the previously identified mutant brevipedicle （bp）. Molecular and genetic analyses showed that the mutant is allelic to bp and that the T-DNA is located within the first helix of the KNAT1 homeodomain （HD）. Although the mutation causes a typical abnormality of short pedicles, propendent siliques, and semidwarfism, no obvious defects are observed in the vegetative stage. A study on cell morphology showed that asymmetrical division and inhibition of cell elongation contribute to the downward-pointing and shorter pedicle phenotype. Loss of KNAT/BPfunction results in the abnormal development of abscission zones. Mlcroarray analysis of gene expression profiling suggests that KNAT1/BP may regulate abscission zone development through hormone signaling and hormone metabolism in Arabidopsis.     

细胞周期后期促进因子DIG9对植物侧根发生的调控研究

在植物体内,细胞周期对于植物的萌发、生长、开花、结实等各个生长发育阶段具有重要作用。细胞周期正常运转需要依赖一些细胞周期蛋白,但是目前关于细胞周期蛋白调控根发育的分子机制还不清楚。通过筛选模式植物拟南芥的根发育异常突变体,分离鉴定了1个突变体dig9（drought inhibition of lateral root growth）,该突变体表现为主根短、侧根少、发育迟缓、顶端分生组织变小、叶片扭曲、无主茎等表型。通过图位克隆,成功定位并克隆了DIG9基因,该基因编码一个细胞周期蛋白,是有丝分裂后期促进复合体的一个亚基APC8 （anaphase-promoting complex）。通过亚细胞定位发现DIG9定位于细胞核;qRT-PCR检测发现DIG9基因在根中有较高的表达量,进一步通过启动子-GUS报告系统发现DIG9在根尖、侧根和顶端分生组织等细胞分裂旺盛区域表达。外施IAA能恢复dig9突变体的侧根表型但不能恢复根短表型。dig9突变体对干旱及盐胁迫反应不敏感。研究结果表明DIG9基因可能通过影响IAA的产生来调控植物的侧根发育。     

Identification and Mapping of a T-DNA Induced Flower Mutation in Arabidopsis Thaliana

Collection of the T-DNA tagged lines of Arabidopsis thaliana have been created by Agrobacterium-mediated root transformation. Transgenic lines produced by this method have been screened for morphogenic mutations. A flower mutation with increased number of stamens and carpels (scaf1) was identified. This mutation has similar but weaker phenotype than the known mutant superman. Two mapping procedures, with visible and molecular markers, were used to locate scaf1 flower mutation. Genetic analysis showed that this mutation is located on chromosome 3 near gl1 gene. It is probably one of the SUPERMAN epigenetic alleles. This revised version was published online in June 2006 with corrections to the Cover Date.     

低氮下拟南芥叶酰聚谷氨酸合成酶突变体atdfb根发育研究

利用叶酰聚谷氨酸合成酶功能缺失突变体atdfb解析叶酸在拟南芥根发育过程中的生物学功能。纯合T-DNA插入功能缺失突变体atdfb 在土壤培养条件下生长3周,与野生型表型无明显差异。在氮源充足的1/2MS培养基上,atdfb的主根显著短于野生型,互补植株的主根长度恢复到野生型水平,说明主根缩短的表型是由AtDFB基因功能缺失造成的。在1/2MS培养基生长11 d的突变体主根长度只有野生型的23%。在低氮条件下,突变体的生长发育几乎停滞,培养11 d的突变体主根长度只有野生型的4%;5-甲酰四氢叶酸(5-F-THF)可以恢复低氮条件下atdfb-3的表型,其主根长度、根毛长度及静止中心的细胞排列均得到恢复。进一步分析发现,低氮条件下培养少于3 d的atdfb-3补充充足的5-F-THF,3 d后能像野生型一样适应低氮环境。由此说明叶酸对拟南芥根部发育及对低氮环境的适应是必需的。     

The COW1 locus of arabidopsis acts after RHD2, and in parallel with RHD3 and TIP1, to determine the shape, rate of elongation, and number of root hairs produced from each site of hair formation.

Two recessive mutant alleles at CAN OF WORMS1 (COW1), a new locus involved in root hair morphogenesis, have been identified in Arabidopsis thaliana L. Heynh. Root hairs on Cow1- mutants are short and wide and occasionally formed as pairs at a single site of hair formation. The COW1 locus maps to chromosome 4. Root hairs on Cow1- plants form in the usual positions, suggesting that the phenotype is not the result of abnormal positional signals. Root hairs on Cow1- roots begin hair formation normally, forming a small bulge, or root hair initiation site, of normal size and shape and in the usual position on the hair-forming cell. However, when Cow1- root hairs start to elongate by tip growth, abnormalities in the shape and elongation rate of the hairs become apparent. Genetic evidence from double-mutant analysis of cow1-1 and other loci involved in root hair development supports our conclusion that COW1 is required during root hair elongation.     

The hpa1 mutant of Arabidopsis reveals a crucial role of histidine homeostasis in root meristem maintenance

Histidine (His) is an essential ingredient for protein synthesis and is required by all living organisms. In higher plants, although there is considerable evidence that His is essential for plant growth and survival, there is very little information as to whether it plays any specific role in plant development. Here, we present evidence for such a role of this amino acid in root development in Arabidopsis (Arabidopsis thaliana) from the characterization of a novel Arabidopsis mutant, hpa1, which has a very short root system and carries a mutation in one of the two Arabidopsis histidinol-phosphate aminotransferase (HPA) genes, AtHPA1. We have established that AtHPA1 encodes a functional HPA and that its complete knockout is embryo lethal. Biochemical analysis shows that the mutation in hpa1 only resulted in a 30% reduction in free His content and had no significant impact on the total His content. It did not cause any known symptoms of His starvation. However, the mutant displayed a specific developmental defect in root meristem maintenance and was unable to sustain primary root growth 2 d after germination. We have demonstrated that the root meristem failure in the mutant is tightly linked to the reduction in free His content and could be rescued by either exogenous His supplementation or AtHPA1 overexpression. Our results therefore reveal an important role of His homeostasis in plant development.