The ACR4 receptor-like kinase is required for surface formation of epidermis-related tissues in Arabidopsis thaliana

In higher plants, an outer layer of meristematic cells, the protoderm, forms early in embryogenesis and this layer gives rise to the epidermis in differentiating tissues. We proposed previously that an Arabidopsis thaliana homolog of crinkly4 (ACR4), a gene for a receptor-like protein kinase, would be involved in differentiation and/or maintenance of epidermis-related tissues. In the present study, we isolated loss-of-function acr4 mutants by a reverse genetic approach. Our extensive analyses using the transmission electron microscopy and the toluidine blue test -- a method that has recently been developed for the rapid visualization of defects in the leaf cuticle -- showed that the acr4 mutations significantly affected the differentiation of leaf epidermal cells, suggesting similar roles for ACR4 and CR4 in the differentiation of leaf epidermis. Our acr4 mutants also had various abnormalities related to epidermal differentiation, which included disorganized cell layers in the integument and endothelium of ovules. In addition, the green fluorescent protein fused to ACR4 was localized preferentially on the lateral and basal plasma membranes in the epidermis of the leaf primordia, suggesting a role for ACR4 in epidermal differentiation at cell surfaces that make contact with adjacent cells. Furthermore, the loss-of-function mutations in the ACR4 and ABNORMAL LEAF SHAPE1 (ALE1) genes, which encode a putative subtilisin-like serine protease, synergistically affected the function of the epidermis such that most leaves fused. Thus, ACR4 seems to play an essential role in the differentiation of proper epidermal cells in both vegetative and reproductive tissues.     

ERECTA receptor-like kinase and heterotrimeric G protein from Arabidopsis are required for resistance to the necrotrophic fungus Plectosphaerella cucumerina

Arabidopsis resistance to the necrotrophic fungus Plectosphaerella cucumerina is complex and depends on the ethylene, jasmonic acid and salicylic acid signaling pathways. A quantitative trait loci (QTL) analysis of resistance to this fungus was performed using two populations of recombinant inbred lines. Three loci QRP1-QRP3 (for Quantitative Resistance to Plectosphaerella) were identified and mapped on chromosome 2 (QRP1 and QRP2) and 5 (QRP3). QRP1, the locus showing the strongest effect, was found to correspond to the ERECTA (ER) gene that encodes a receptor-like-kinase (RLK), which has been previously implicated in plant development, and resistance to the bacterium Ralstonia solanacearum. The leucine-rich repeat and the kinase domains of ERECTA were specifically required for resistance to P. cucumerina, as er mutant alleles impaired in any of these domains showed enhanced susceptibility to this fungus, but not to other virulent pathogens. The involvement of the ER-signaling pathway in resistance to P. cucumerina was supported by the fact that three mutants defective in this pathway, elk2, elk5 and elk4 (agb1-1), which encodes the beta-subunit of Arabidopsis heterotrimeric G protein, were also impaired in their resistance to this fungus. The putative function of the Arabidopsis heterotrimeric G protein in resistance to P. cucumerina suggested by the enhanced susceptibility of agb1-1 was corroborated by the demonstration that a null allele (gpa1-4) of the G protein alpha-subunit showed enhanced resistance to this pathogen. Deposition of beta-1,3-glucan callose at infection sites was specifically impaired in er-1 and agb1-1 mutants upon P. cucumerina inoculation. Taken together, these data suggest a putative function of ERECTA and heterotrimeric G protein in P. cucumerina perception.     

植物凝集素类受体激酶参与非生物胁迫响应的研究进展

植物在生长发育过程中会受到各种胁迫因子的影响,非生物胁迫是其中极其重要的一类。类受体激酶(receptor-like kinases, RLKs)是植物中广泛存在的一类蛋白,能够快速有效地对胁迫因子作出响应,最终引起一系列生物效应。凝集素类受体激酶(lectin receptor-like kinases, LecRLKs)是RLKs的一个亚族,其具有细胞外凝集素结构域、跨膜结构域和细胞内激酶结构域三个结构域。根据细胞外凝集素结构域的不同可分为L、G和C三种不同类型。近年来,大量的研究表明植物凝集素类受体激酶在非生物胁迫响应中发挥重要作用。LecRLKs通过识别非生物胁迫相关的信号分子,激活下游的信号通路,如MAPK通路、ROS通路、钙信号通路等,调节基因表达和蛋白质翻译以增强植物的抗逆性。该文概述了植物凝集素类受体激酶的结构特征及其分类,并系统综述了LecRLKs在盐胁迫、低温胁迫、干旱胁迫、机械损伤和植物激素等非生物胁迫响应中的功能和作用机制,同时也对LecRLKs的未来研究方向作出了展望。该文不仅为深入了解植物凝集素类受体激酶参与非生物胁迫响应的功能提供了参考,而且为利用LecRLKs进行作物抗逆育种改良提供了理论依据。     

OsLTP47 may function in a lipid transfer relay essential for pollen wall development in rice

In plants, lipid transfer proteins(LTPs) transport pollen wall constituents from the tapetum to the exine, a process essential for pollen wall development. However, the functional cooperation of different LTPs in pollen wall development is not well understood. In this study, we have identified and characterized a grassspecific LTP gene, Os LTP47, an important regulator of pollen wall formation in rice(Oryza sativa). Os LTP47 encodes a membrane-localized LTP and in vitro lipid-binding assays conf...     

Chemical agents that inhibit pollen development: tools for research

Summary Several unrelated compounds are known to selectively inhibit the development of the male gametophyte. When applied at suitable dosages to plants at the appropriate stages of anther development, these substances block the formation of fertile pollen. The affected stage of pollen development is characteristic of the specific chemical structure of the compound, ranging from effects on microspore meiosis to the formation of pollen defective in the ability to germinate or fertilize. The range of effects mediated by these substances, and by known male-sterile mutants, indicates that microspore development has several critical phases that are particularly sensitive to fatal inhibition. We propose that chemical inhibitors of pollen development deserve attention as tools for elucidating the regulation of pollen development.     

A Nicotiana tabacum mRNA encoding a 69-kDa glycoprotein occurring abundantly in pollen tubes is transcribed but not translated during pollen development in the anthers

Regulation of expression of a 69-kDa glycoprotein which occurs abundantly in tobacco (Nicotiana tabacum L.) pollen tubes but is absent in ungerminated pollen has been studied in vitro by means of a coupled translation/glycosylation system with RNA isolated from various stages of pollen development. Pollen mRNA could be translated in a rabbit reticulocyte lysate and the products glycosylated with canine pancreatic microsomal membranes. The electrophoretic pattern of translation products obtained with pollen-tube RNA showed a prominent polypeptide with an apparent molecular mass of 58 kDa. In the presence of the canine pancreatic microsomal membranes this polypeptide was glycosylated, producing the 69-kDa glycoprotein. The presence of mRNA encoding the 58-kDa precursor polypeptide was also demonstrated in ungerminated pollen and in young mid-binucleate pollen isolated from anthers. Initiation of synthesis of the 69-kDa glycoprotein at the onset of pollen germination thus occurs through unmasking of the mRNA transcribed during pollen differentiation and stored during pollen maturation and dormancy in an inactive state.Abbreviation pI isoelectric point     

Involvement of phenylalanine ammonia-lyase in the development of pollen in broccoli (Brassica oleracea L.)

Summary To determine whether phenylalanine ammonia-lyase (EC 4.3.1.5) is involved in the maturation of microspores to fertile pollen, anthers of a fertile strain of broccoli (Brassica oleracea L.) were studied in a comparison with anthers of a cytoplasmic male sterile strain. In the normal fertile strain, immature anthers of about 2 mm in length exhibited higher phenylalanine ammonia-lyase activity than mature anthers or those shorter than 2 mm. The 2-mm-long anthers corresponded to the mononucleate stage, just after release of the microspores during pollen development. Immunohistochemical localization of phenylalanine ammonia-lyase in the anthers indicated that the protein was present predominantly in the tapetal cells. The immature anthers of cytoplasmic male sterile broccoli had a lower phenylalanine ammonia-lyase activity than those of the normal fertile strain. The level of phenylalanine ammonia-lyase activity in the immature anthers was positively correlated with the number of fertile pollen grains at the flowering stage in both strains. It seems possible, therefore, that phenylpropanoid metabolism, which involves phenylalanine ammonia-lyase, may play an important role in the maturation of microspores in flowering plants.Abbreviations CHS chalcone synthase - CMS cytoplasmic male sterility - DAPI 4, 6-diamidmo-2-phenylindole dihydrochloride - PAL L-phenylalanine ammonia-lyase     

Analysis of gene expression profile in pollen development of recessive genic male sterile <Emphasis Type="Italic">Brassica napus</Emphasis> L. line S45A

Male sterility in a near-isogenic line S45AB after 25 generations of subcrossing is controlled by two pairs of duplicate genes. The genotype of S45A is Bnms1Bnms1Bnms2Bnms2, and that of S45B is BnMs1Bnms1Bnms2Bnms2, respectively. Histological observations revealed that abnormal anther development appeared in the tapetum and pollen exine during the tetrad stage. This male sterility was characterized by hypertrophy of the tapetal cells at the tetrad stage and a complete lack of microspore exine after the release of microspores from the tetrads. To elucidate the mechanism of this recessive genic male sterility, the flower bud expression profiles of the S45A and S45B lines were analyzed using an Arabidopsis thaliana ATH1 oligonucleotide array. When compared with the S45B line, 69 genes were significantly downregulated, and 46 genes were significantly upregulated in the S45A line. Real-time polymerase chain reaction (PCR) was then used to verify the results of the microarray analysis, and the majority of the downregulated genes in the S45A line were abundantly and specifically expressed in the anther. The results of the real-time PCR suggest that Bnms1 might be involved in the metabolism of lipid/fatty acids, and the homologous mutation of Bnms1 may either block the biosynthesis of sporopollenin or block sporopollenin from being deposited on the microspore surface, thus, preventing pollen exine formation. The role of Bnms1 in the regulatory network of exine formation is also discussed as well. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In vitro pollen embryogenesis in Nicotiana tabacum L. and its relation to pollen sterility,sex balance,and floral induction of the pollen donor plants

Pollen sterility, sex balance, and floral induction of the pollen donor plants were tested for a possible relation to embryogenesis from in vitro cultured tobacco pollen (Nicotiana tabacum L. var. Badischer Burley). The pollen grains destined to become embryos in culture (P-grains) were sterile for the donor plants as judged by their staining reaction with acetocarmine and fluorescin-diacetate, and by an in vitro germination test. They were produced in high frequency in flowers which exhibited a shift in sex balance towards femaleness. Sex balance could be measured by the relative length of pistil to stamens. High P-grain frequency, high pollen sterility, and a shift in sex balance towards femaleness could be induced by raising the donor plants under short days and/or low temperature (18–15° C) as compared to long days at 24° C. Short days and/or low temperature also reinforced floral induction, revealing that the tobacco variety Badischer Burley is a quantitative short day and low temperature plant and that the variety follows the rule that conditions of strong floral induction shift sex balance towards femaleness. At 12° C and short days, contabescent flowers were formed with completely sterile anthers containing a few and mostly collapsed P-grains. Based on these results, it is now possible to predict conditions by which haploids via pollen embryogenesis might be produced in high frequency from low-yielding and recalcitrant species.Abbreviations DPF dead pollen grain frequency - LD24 long days at 24° C - PD pollen dimorphism - P:S ratio of pistil to stamen length - SD15 short days at 15° C     

BRL1, a leucine-rich repeat receptor-like protein kinase, is functionally redundant with BRI1 in regulating Arabidopsis brassinosteroid signaling

BRI1-like receptor kinase (BRL1) was identified as an extragenic suppressor of a weak bri1 allele, bri1-5, in an activation-tagging genetic screen for novel brassinosteroid (BR) signal transduction regulators. BRL1 encodes a leucine-rich repeat receptor-like protein kinase (LRR-RLK). Sequence alignment revealed that BRL1 is closely related to BRI1, which is involved in BR perception. Overexpression of a BRL1 cDNA, driven by a constitutive CaMV 35S promoter, recapitulates the bri1-5 suppression phenotypes, and partially complements the phenotypes of a null bri1 allele, bri1-4. Analysis of a BR-specific feedback response gene, CPD, indicates that BRL1 functions in BR signaling. BRL1 expression pattern overlaps with, but is distinct from, that of BRI1. In addition, both the expression level and in vitro kinase autophosphorylation activity of BRL1 are significantly lower than those of BRI1. bri1-5 brl1-1 double mutant plants have enhanced developmental defects relative to bri1-5 mutant plants, revealing that BRL1 plays a partially redundant role with BRI1 in controlling Arabidopsis growth and development. These findings enhance our understanding of functional redundancy and add an additional layer of complexity to RLK-mediated BR signaling transduction in Arabidopsis.     

Callose synthase (CalS5) is required for exine formation during microgametogenesis and for pollen viability in Arabidopsis

Callose (beta-1,3-glucan) is produced at different locations in response to biotic and abiotic cues. Arabidopsis contains 12 genes encoding callose synthase (CalS). We demonstrate that one of these genes, CalS5, encodes a callose synthase which is responsible for the synthesis of callose deposited at the primary cell wall of meiocytes, tetrads and microspores, and the expression of this gene is essential for exine formation in pollen wall. CalS5 encodes a transmembrane protein of 1923 amino acid residues with a molecular mass of 220 kDa. Knockout mutations of the CalS5 gene by T-DNA insertion resulted in a severe reduction in fertility. The reduced fertility in the cals5 mutants is attributed to the degeneration of microspores. However, megagametogenesis is not affected and the female gametes are completely fertile in cals5 mutants. The CalS5 gene is also expressed in other organs with the highest expression in meiocytes, tetrads, microspores and mature pollen. Callose deposition in the cals5 mutant was nearly completely lacking, suggesting that this gene is essential for the synthesis of callose in these tissues. As a result, the pollen exine wall was not formed properly, affecting the baculae and tectum structure and tryphine was deposited randomly as globular structures. These data suggest that callose synthesis has a vital function in building a properly sculpted exine, the integrity of which is essential for pollen viability.     

The HKM gene, which is identical to the MS1 gene of Arabidopsis thaliana, is essential for primexine formation and exine pattern formation

A male-sterile mutant of Arabidopsis thaliana was isolated by T-DNA tagging screening. Using transmission electron microscopy analysis, we revealed that the microspores of this mutant did not have normal thick primexine on the microspore at the tetrad stage. Instead, a moderately electron-dense layer formed around the microspores. Although microspores without normal primexine failed to form a proper reticulate exine pattern at later stages, sporopollenin was deposited and an exine-like hackly structure was observed on the microspores during the microspore stage. Thus, this mutant was named hackly microspore (hkm). It is speculated that the moderately electron-dense layer was primexine, which partially played its role in sporopollenin deposition onto the microspore. Cytological analysis revealed that the tapetum of the hkm mutant was significantly vacuolated, and that vacuolated tapetal cells crushed the microspores, resulting in the absence of pollen grains within the anther at anthesis. Single nucleotide polymorphism analysis demonstrated that the hkm mutation exists within the MS1 gene, which has been reportedly expressed within the tapetum. Our results suggest that the critical process of primexine formation is under sporophytic control .     

小立碗藓LysM型类受体激酶基因家族生物信息学分析

植物LysM型类受体激酶(lysin motif receptor-like kinase,LYKs)是植物中发现的一类重要的RLK,在植物生长发育、抵御逆境胁迫等方面具有不可忽视的作用,是植物中基因功能的研究热点.为更好地了解小立碗藓中的LYK基因,该文利用生物信息学的方法对小立碗藓(Physcomitrella p...     

Thiamin pyrophosphokinase is required for thiamin cofactor activation in Arabidopsis

Thiamin pyrophosphate (TPP) is an essential enzyme cofactor required for the viability of all organisms. Whether derived from exogenous sources or through de novo synthesis, thiamin must be pyrophosphorylated for cofactor activation. The enzyme thiamin pyrophosphokinase (TPK) catalyzes the conversion of free thiamin to TPP in plants and other eukaryotic organisms and is central to thiamin cofactor activation. While TPK activity has been observed in a number of plant species, the corresponding gene/protein has until now not been identified or characterized for its role in thiamin metabolism. Here we report the functional identification of two Arabidopsis TPK genes, AtTPK1 and AtTPK2 and the enzymatic characterization of the corresponding proteins. AtTPK1 and AtTPK2 are biochemically redundant cytosolic proteins that are similarly expressed throughout different plant tissues. The essential nature of TPKs in plant metabolism is reflected in the observation that while single gene knockouts of either AtTPK1 or AtTPK2 were viable, the double mutant possessed a seedling lethal phenotype. HPLC analysis revealed the double mutant is nearly devoid of TPP and instead accumulates the precursor of the TPK reaction, free thiamin. These results suggest that TPK activity provides the sole mechanism by which exogenous and de novo derived thiamin is converted to the enzyme cofactor TPP.