The moss genes <Emphasis Type="Italic">PpSKI1</Emphasis> and <Emphasis Type="Italic">PpSKI2</Emphasis> encode nuclear SnRK1 interacting proteins with homologues in vascular plants

The yeast Snf1, animal AMPK, and plant SnRK1 protein kinases constitute a family of related proteins that have been proposed to serve as metabolic sensors of the eukaryotic cell. We have previously reported the characterization of two redundant SnRK1 encoding genes (PpSNF1a and PpSNF1b) in the moss Physcomitrella patens. Phenotypic analysis of the snf1a snf1b double knockout mutant suggested that SnRK1 is important for the plant’s ability to recognize and adapt to conditions of limited energy supply, and also suggested a possible role of SnRK1 in the control of plant development. We have now used a yeast two-hybrid system to screen for PpSnf1a interacting proteins. Two new moss genes were found, PpSKI1 and PpSKI2, which encode highly similar proteins with homologues in vascular plants. Fusions of the two encoded proteins to the green fluorescent protein localize to the nucleus. Knockout mutants for either gene have an excess of gametophores under low light conditions, and exhibit reduced gametophore stem lengths. Possible functions of the new proteins and their connection to the SnRK1 kinase are discussed.     

Cloning of the PpMSH-2 cDNA of Physcomitrella patens, a moss in which gene targeting by homologous recombination occurs at high frequency

In the moss Physcomitrella patens integrative transformants from homologous recombination are obtained at an efficiency comparable to that found for yeast. This property, unique in the plant kingdom, allows the knockout of specific genes. It also makes the moss a convenient model to study the regulation of homologous recombination in plants. We used degenerate oligonucleotides designed from AtMSH2 from Arabidopsis thaliana and other known MutS homologues to isolate the P. patens MSH2 (PpMSH2) cDNA. The deduced sequence of the PpMSH2 protein is respectively 60.8% and 59.6% identical to the maize and A. thaliana MSH2. Phylogenic studies show that PpMSH2 is closely related to the group of plant MSH2 proteins. Southern analysis reveals that the gene exists as a single copy in the P. patens genome.     

Revelation of ancestral roles of KNOX genes by a functional analysis of <Emphasis Type="Italic">Physcomitrella</Emphasis> homologues

KNOX genes are indispensable elements of indeterminate apical growth programmes of vascular plant sporophytes. Since little is known about the roles of such genes in non-vascular plants, functional analysis of moss KNOX homologues (MKN genes) was undertaken using the genetically amenable model plant, Physcomitrella patens. Three MKN genes were inactivated by targeted gene knockout to produce single, double and triple mutants. MKN2 (a class 1 KNOX gene) mutants were characterised by premature sporogenesis, abnormal sporophyte ontogeny and irregular spore development. MKN4 (a second class 1 gene) mutants were phenotypically normal. MKN1-3 (a class 2 KNOX gene) mutants exhibited defects in spore coat morphology. Analysis of double and triple mutants revealed that the abnormal sporophytic phenotype of MKN2 mutants was accentuated by mutating MKN4 and to a lesser degree by mutating MKN1-3. The aberrant spore phenotype of MKN1-3 and MKN2 mutants was exacerbated by mutating MKN4. This study provides the first instance in which an abnormal phenotype has been associated with the disruption of a class 2 KNOX gene as well as the first demonstrated case of functional redundancy between a class 1 and a class 2 KNOX gene. We conclude that KNOX genes play significant roles in programming sporophytic development in moss and we provide evidence that ancestral function(s) of this gene family were instrumental in the successful transition of plants to a terrestrial environment.     

Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens

The blue light receptors termed cryptochromes mediate photomorphological responses in seed plants. However, the mechanisms by which cryptochrome signals regulate plant development remain obscure. In this study, cryptochrome functions were analyzed using the moss Physcomitrella patens. This moss has recently become known as the only plant species in which gene replacement occurs at a high frequency by homologous recombination. Two cryptochrome genes were identified in Physcomitrella, and single and double disruptants of these genes were generated. Using these disruptants, it was revealed that cryptochrome signals regulate many steps in moss development, including induction of side branching on protonema and gametophore induction and development. In addition, the disruption of cryptochromes altered auxin responses, including the expression of auxin-inducible genes. Cryptochrome disruptants were more sensitive to external auxin than wild type in a blue light-specific manner, suggesting that cryptochrome light signals repress auxin signals to control plant development.     

Glyco-engineering of moss lacking plant-specific sugar residues

The commercial production of complex pharmaceutical proteins from human origin in plants is currently limited through differences in protein N-glycosylation pattern between plants and humans. On the one hand, plant-specific alpha(1,3)-fucose and beta(1,2)-xylose residues were shown to bear strong immunogenic potential. On the other hand, terminal beta(1,4)-galactose, a sugar common on N-glycans of pharmaceutically relevant proteins, e.g., antibodies, is missing in plant N-glycan structures. For safe and flexible production of pharmaceutical proteins, the humanisation of plant protein N-glycosylation is essential. Here, we present an approach that combines avoidance of plant-specific and introduction of human glycan structures. Transgenic strains of the moss Physcomitrella patens were created in which the alpha(1,3)-fucosyltransferase and beta(1,2)-xylosyltransferase genes were knocked out by targeted insertion of the human beta(1,4)-galactosyltransferase coding sequence in both of the plant genes (knockin). The transgenics lacked alpha(1,3)-fucose and beta(1,2)-xylose residues, whereas beta(1,4)-galactose residues appeared on protein N-glycans. Despite these significant biochemical changes, the plants did not differ from wild type with regard to overall morphology under standard cultivation conditions. Furthermore, the glyco-engineered plants secreted a transiently expressed recombinant human protein, the vascular endothelial growth factor, in the same concentration as unmodified moss, indicating that the performed changes in glycosylation did not impair the secretory pathway of the moss. The combined knockout/knockin approach presented here, leads to a new generation of engineered moss and towards the safe and flexible production of correctly processed pharmaceutical proteins with humanised N-glycosylation profiles.     

Snf1-related protein kinase 1 is needed for growth in a normal day-night light cycle

The yeast Snf1 protein kinase and its animal homologue, the AMP-activated protein kinase, play important roles in metabolic regulation, by serving as energy gauges that turn off energy-consuming processes and mobilize energy reserves during low-energy conditions. The closest homologue of these kinases in plants is Snf1-related protein kinase 1 (SnRK1). We have cloned two SnRK1-encoding genes, PpSNF1a and PpSNF1b, in the moss Physcomitrella patens, where gene function can be studied directly by gene targeting in the haploid gametophyte. A snf1a snf1b double knockout mutant is viable, but lacks all Snf1-like protein kinase activity. The mutant has a complex phenotype that includes developmental abnormalities, premature senescence and altered sensitivities to plant hormones. Remarkably, the double knockout mutant also requires continuous light, and is unable to grow in a normal day-night light cycle. This suggests that SnRK1 is needed for metabolic changes that help the plant cope with the dark hours of the night.     

海岛棉GbHCT13基因的功能验证

该研究利用海岛棉‘新海21’和陆地棉ND203以及模式植物拟南芥,通过转基因及荧光定量检测等方法探究海岛棉GbHCT13基因(GenBank 登录号MW048849)在纤维发育中的功能。结果显示：(1)成功构建重组载体pCAMBIA3301 GbHCT13,经农杆菌介导法转化、除草剂抗性基因筛选、荧光定量检测方法鉴定获得转GbHCT13基因拟南芥T₃代植株4株;qRT PCR检测表明,转基因植株中GbHCT13基因表达量较野生型极显著增加。(2)转基因拟南芥过表达GbHCT13基因使植株同一时期的生长较野生型旺盛,株形、叶片数、抽薹数和茎秆表皮毛数量均与野生型存在差异;组织化学分析发现,转GbHCT13基因的拟南芥较野生型茎秆初生木质部生长活跃,导管增粗,次生木质部导管细胞壁横截面积变大,但髓质细胞无明显变化;过表达GbHCT13使拟南芥中木质素合成途径基因发生不同程度改变,其中CAD、CCoAOMT、PAL和4CL与GbHCT13基因的表达呈正相关。(3)经大田筛选、分子鉴定,成功获得转GbHCT13基因棉花植株3株;转GbHCT13基因棉花的棉纤维伸长率增加,纤维强度增大;沉默GbHCT13基因使棉花植株木质素含量降低,茎秆表皮毛数量减少,木质部导管细胞数量减少,导管细胞壁中木质素沉积量降低,而棉株并未发生株高上的明显矮化现象,且木质素合成通路中的CAD、CCoAOMT、CCR、PAL 4个基因的表达均呈降低趋势,说明抑制GbHCT13使得棉花生长代谢受阻,影响纤维发育起始。研究表明,GbHCT13基因能影响棉花植株中木质素合成从而调控纤维的生长发育,其功能与GbHCT13基因在模式植物拟南芥中的基本一致。     

Overexpression of a NTR1 in transgenic soybean confers tolerance to water stress

Methyl jasmonate (MeJA) is an important plant regulator that involves in plant development and regulates the expression of plant defense genes in response to various stresses such as wounding, drought, and pathogens. In order to determine the physiological role of endogenous MeJA in plants, a NTR1 from Brassica campestris encoding a jasmonic acid carboxyl methyltransferase that produces methyl jasmonate was constructed under the control of CaMV 35S promoter and transformed into soybean [Glycine max (L) Merrill]. The transgenic soybean plants constitutively expressed the NTR1 and accumulated more MeJA levels than wild type plants. Overexpression of the gene in transgenic soybean conferred tolerance to dehydration during seed germination and seedling growth as reflected by the percentage of the fresh weight of seedlings. In addition, the transgenic soybean plants also conferred better capacity to retain water than wild type plants when drought tolerance was tested using detached leaves.     

A small family of LLS1-related non-heme oxygenases in plants with an origin amongst oxygenic photosynthesizers

Conservation of Lethal-leaf spot 1 (Lls1) lesion mimic gene in land plants including moss is consistent with its recently reported function as pheophorbide a oxygenase (Pao) which catalyzes a key step in chlorophyll degradation (Pruzinska et al., 2003). A bioinformatics survey of complete plant genomes reveals that LLS1(PAO) belongs to a small 5-member family of non-heme oxygenases defined by the presence of Rieske and mononuclear iron-binding domains. This gene family includes chlorophyll a oxygenase (Cao), choline monooxygenase (Cmo), the gene for a 55 kDa protein associated with protein transport through the inner chloroplast membrane (Tic 55) and a novel 52 kDa protein isolated from chloroplasts (Ptc 52). Analysis of gene structure reveals that these genes diverged prior to monocot/dicot divergence. Homologues of LLS1(PAO), CAO, TIC55 and PTC52 but not CMO are found in the genomes of several cyanobacteria. LLS1(PAO), PTC52, TIC55 and a set of related cyanobacterial homologues share an extended carboxyl terminus containing a novel F/Y/W-x(2)-H-x(3)-C-x(2)-C motif not present in CAO. These proteins appear to have evolved during the transition to oxygenic photosynthesis to play various roles in chlorophyll metabolism. In contrast, CMO homologues are found only in plants and are most closely related to aromatic ring-hydroxylating enzymes from soil-dwelling bacteria, suggesting a more recent evolution of this enzyme, possibly by horizontal gene transfer. Our phylogenetic analysis of 95 extant non-heme dioxygenases provides a useful framework for the classification of LLS1(PAO)-related non-heme oxygenases.     

Toc64 is not required for import of proteins into chloroplasts in the moss Physcomitrella patens

Toc64 has been suggested to be part of the chloroplast import machinery in Pisum sativum. A role for Toc64 in protein transport has not been established, however. To address this, we generated knockout mutants in the moss Physcomitrella patens using the moss's ability to perform homologous recombination with nuclear DNA. Physcomitrella patens contains two genes that encode Toc64-like proteins. Both of those proteins appear to be localized in the chloroplast. The double-mutant plants were lacking Toc64 protein in the chloroplasts but showed no growth phenotype. In addition, these plants accumulated other plastid proteins at wild-type levels and showed no difference from wild type in in vitro protein import assays. These plants did have a slightly altered chloroplast shape in some tissues, however. The evidence therefore indicates that Toc64 proteins are not required for import of proteins in Physcomitrella, but may point to involvement in the determination of plastid shape.     

A description of the Mei2-like protein family; structure,phylogenetic distribution and biological context

The Schizosaccharomyces pombe Mei2 gene encodes an RNA recognition motif (RRM) protein that stimulates meiosis upon binding a specific non-coding RNA and subsequent accumulation in a "mei2-dot" in the nucleus. We present here the first systematic characterization of the family of proteins with characteristic Mei2-like amino acid sequences. Mei2-like proteins are an ancient eukaryotic protein family with three identifiable RRMs. The C-terminal RRM (RRM3) is unique to Mei2-like proteins and is the most highly conserved of the three RRMs. RRM3 also contains conserved sequence elements at its C-terminus not found in other RRM domains. Single copy Mei2-like genes are present in some fungi, in alveolates such as Paramecium and in the early branching eukaryote Entamoeba histolytica, while plants contain small families of Mei2-like genes. While the C-terminal RRM is highly conserved between plants and fungi, indicating conservation of molecular mechanisms, plant Mei2-like genes have changed biological context to regulate various aspects of developmental pattern formation.     

Clues about the ancestral roles of plant MADS-box genes from a functional analysis of moss homologues

Classic MIKC-type MADS-box genes (MIKC ^c genes) are indispensable elements in the genetic programming of pattern formation, including the segmental organisation of angiosperm flowers, in seed plants. Since little is known about the functions of MIKC ^c genes in non-seed plants, a functional analysis of moss MIKC ^c homologues was performed using the genetically amenable, simple model plant, Physcomitrella patens. Expression of moss homologues was knocked down using an antisense RNA approach or abolished by generating transformants with gene knockouts. The knocked down (“antisense”) transformants displayed a multifaceted mutant phenotype comprising delayed gametangia formation, diminished sporophyte yield and, in the most extremely affected cases, abnormal sporophyte development and altered leaf morphogenesis. Knocked out transformants were phenotypically normal. Analysis of in situ MIKC ^c gene expression using transgenic strains containing MIKC ^c promoter–GUS fusions showed that these genes are generally expressed ubiquitously in vegetative and reproductive tissues. We conclude that MIKC ^c genes play significant roles in morphogenetic programming of the moss. Functional redundancy characterises some members of the gene group. Our findings provide clues concerning the ancestral roles of some MIKC ^c genes that may be represented in the genomes of diverse extant plant taxa. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The PsbQ protein is required in Arabidopsis for photosystem II assembly/stability and photoautotrophy under low light conditions

RNA interference was used to simultaneously suppress the expression of the two genes that encode the PsbQ proteins of Photosystem II (PS II) in Arabidopsis thaliana, psbQ-1 (At4g21280) and psbQ-2 (At4g05180). Two independent PsbQ-deficient plant lines were examined. These plant lines produced little detectable PsbQ protein. Under normal growth light conditions, the wild type and mutant plants were visually indistinguishable. Additionally, analysis of steady state oxygen evolution rates and chlorophyll fluorescence characteristics indicated little alteration of photosynthetic capacity in the mutant plants. No loss of other PS II proteins was evident. Interestingly, flash oxygen yield analysis performed on thylakoid membranes isolated from the mutant and wild type plants indicated that the oxygen-evolving complex was quite unstable in the mutants. Furthermore, the lifetime of the S2 state of the oxygen-evolving complex appeared to be increased in these plants. Incubation of the wild type and mutant plants under low light growth conditions led to a significantly stronger observed phenotype in the mutants. The mutant plants progressively yellowed (after 2 weeks) and eventually died (after 3-4 weeks). The wild type plants exhibited only slight yellowing after 4 weeks under low light conditions. The mutant plants exhibited a large loss of a number of PS II components, including CP47 and the D2 protein, under low light conditions. Additionally, significant alterations of their fluorescence characteristics were observed, including an increased FO and decreased FV, yielding a large loss in PS II quantum efficiency (FV/FM). Analysis of QA- decay kinetics in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea indicated a defect in electron transfer from QA- to QB, whereas experiments performed in the presence of this herbicide indicated that the recombination rate between QA- and the S2 state was strongly retarded. These results indicate that the loss of the PsbQ protein induces significant changes in Photosystem II function, particularly in low light-grown plants, and that the PsbQ protein is required for photoautotrophic growth under low light conditions.