Digalactosyldiacylglycerol is required for better photosynthetic growth of Synechocystis sp. PCC6803 under phosphate limitation

Digalactosyldiacylglycerol (DGDG) is a typical membrane lipid of oxygenic photosynthetic organisms. Although DGDG synthase genes have been isolated from plants, no homologous gene has been annotated in the genomes of cyanobacteria and the unicellular red alga Cyanidioschyzon merolae. Here we used a comparative genomics approach and identified a non-plant-type DGDG synthase gene (designated dgdA) in Synechocystis sp. PCC6803. The enzyme produced DGDG in Escherichia coli when co-expressed with a cucumber monogalactosyldiacylglycerol synthase. A DeltadgdA knock-out mutant showed no obvious phenotype other than loss of DGDG when grown in a BG11 medium, indicating that DGDG is dispensable under optimal conditions. However, the mutant showed reduced growth under phosphate-limited conditions, suggesting that DGDG may be required under phosphate-limited conditions, such as those in natural niches of cyanobacteria.     

Arabidopsis VPS35, a retromer component, is required for vacuolar protein sorting and involved in plant growth and leaf senescence

The retromer complex is responsible for retrograde transport,which is coordinated with anterograde transport in the secretorypathway including vacuolar protein sorting. Yeast VPS35 is acomponent of the retromer complex that is essential for recognitionof specific cargo molecules. The physiological function of VPS35has not been determined in vacuolar protein sorting in higherorganisms. Arabidopsis thaliana has three VPS35 homologs designatedVPS35a, VPS35b and VPS35c. We isolated four vps35 mutants (vps35a-1,vps35b-1, vps35b-2 and vps35c-1) and then generated four doublemutants and one triple mutant. vps35a-1 vps35c-1 exhibited nounusual phenotypes. On the other hand, vps35b-1 vps35c-1 andthe triple mutant (vps35a-1 vps35b-2 vps35c-1) exhibited severephenotypes: dwarfism, early leaf senescence and fragmentationof protein storage vacuoles (PSVs). In addition, these mutantsmis-sorted storage proteins by secreting them out of the cellsand accumulated a higher level of vacuolar sorting receptor(VSR) than the wild type. VPS35 was localized in pre-vacuolarcompartments (PVCs), some of which contained VSR. VPS35 wasimmunoprecipitated with VPS29/MAG1, another component of theretromer complex. Our findings suggest that VPS35, mainly VPS35b,is involved in sorting proteins to PSVs in seeds, possibly byrecycling VSR from PVCs to the Golgi complex, and is also involvedin plant growth and senescence in vegetative organs.     

The FLX gene of Arabidopsis is required for FRI-dependent activation of FLC expression

The Arabidopsis FLOWERING LOCUS C (FLC) gene encodes a MADS box protein that acts as a dose-dependent repressor of flowering. Mutants and ecotypes with elevated expression of FLC are late flowering and vernalization responsive. In this study we describe an early flowering mutant in the C24 ecotype, flc expressor (flx), that has reduced expression of FLC. FLX encodes a protein of unknown function with putative leucine zipper domains. FLX is required for FRIGIDA (FRI)-mediated activation of FLC but not for activation of FLC in autonomous pathway mutants. FLX is also required for expression of the FLC paralogs MADS AFFECTING FLOWERING 1 (MAF1) and MAF2.     

A balanced PGR5 level is required for chloroplast development and optimum operation of cyclic electron transport around photosystem I

PSI cyclic electron transport contributes markedly to photosynthesis and photoprotection in flowering plants. Although the thylakoid protein PGR5 (Proton Gradient Regulation 5) has been shown to be essential for the main route of PSI cyclic electron transport, its exact function remains unclear. In transgenic Arabidopsis plants overaccumulating PGR5 in the thylakoid membrane, chloroplast development was delayed, especially in the cotyledons. Although photosynthetic electron transport was not affected during steady-state photosynthesis, a high level of non-photochemical quenching (NPQ) was transiently induced after a shift of light conditions. This phenotype was explained by elevated activity of PSI cyclic electron transport, which was monitored in an in vitro system using ruptured chloroplasts, and also in leaves. The effect of overaccumulation of PGR5 was specific to the antimycin A-sensitive pathway of PSI cyclic electron transport but not to the NAD(P)H dehydrogenase (NDH) pathway. We propose that a balanced PGR5 level is required for efficient regulation of the rate of antimycin A-sensitive PSI cyclic electron transport, although the rate of PSI cyclic electron transport is probably also regulated by other factors during steady-state photosynthesis.     

Cellulose synthesis is required for deposition of reticulate wall ingrowths in transfer cells

Despite the recognized physiological importance of transfer cells, little is known about how these specialized cells achieve localized deposition of cell wall material, leading to amplification of plasma membrane surface area and enhanced membrane transport capacity. This study establishes that cellulose synthesis is a key early factor in the construction of 'reticulate' wall ingrowths, an elaborate but common form of localized wall deposition characteristic of most transfer cells. Using field emission scanning electron microscopy, wall ingrowths were first visible in epidermal transfer cells of Faba bean cotyledons as raised 'patches' of disorganized and tangled cellulosic material, and, from these structures, ingrowths emerged via further deposition of wall material. The cellulose biosynthesis inhibitors 2,6-dichlorobenzonitrile and isoxaben both caused dramatic reductions in the number of cells depositing wall ingrowths, altered wall ingrowth morphology and visibly disrupted microfibril structure. The restriction of cellulose deposition to discrete patches suggests a novel mechanism for cellulose synthesis in this circumstance. Overall, these results implicate a central role for cellulose synthesis in reticulate wall ingrowth morphology, especially at the initial stage of ingrowth formation, possibly by providing a template for the self-assembly of wall polymers.     

Genetic transformation of Populus trichocarpa genotype Nisqually-1: a functional genomic tool for woody plants

We report here the Agrobacterium-mediated genetic transformation of Nisqually-1, a Populus trichocarpa genotype whose genome was recently sequenced. Several systems were established. Internodal stem segments from vigorously growing greenhouse plants are the explants most amenable to transformation. For the most efficient system, approximately 40% of the stem segments infected with pBI121-containing Agrobacterium tumefaciens C58 produced transgenic calli, as confirmed by beta-glucuronidase (GUS) staining. The regeneration efficiency of independent transgenic plants was approximately 13%, as revealed by genomic Southern analysis. Some transgenic plants were produced in as little as 5 months after co-cultivation. This system may help to facilitate studies of gene functions in tree growth and development at a genome level.     

A rice dihydrosphingosine C4 hydroxylase (DSH1) gene, which is abundantly expressed in the stigmas, vascular cells and apical meristem, may be involved in fertility

Dihydrosphingosine C4 hydroxylase is a key enzyme in the biosynthesis of phytosphingosine, a major constituent of sphingolipids in plants and yeasts. The rice genome contains five homologue genes for dihydrosphingosine C4 hydroxylase, DSH1-DSH5, whose gene products show high degrees of homology to the yeast counterpart, SUR2. Among them, expression of DSH1, DSH2 and DSH4 was detected, and DSH1 and DSH4 complement the yeast sur2 mutation. The DSH1 gene was specifically and abundantly expressed in vascular bundles and apical meristems. In particular, very strong expression was detected in the stigmas of flowers. Repression of DSH1 expression by the antisense gene or RNA interference (RNAi) resulted in a severe reduction of fertility. In the transformants in which DSH1 expression was suppressed, significantly increased expression of DSH2 was found in leaves but not in pistils, suggesting that there was tissue-specific correlation between DSH1 and DSH2 expression. Our results indicate that the product of DSH1 may be involved in plant viability or reproductive processes, and that the phenotype of sterility is apparently caused by loss of function of DSH1 in the stigma. It is also suggested that there is a complex mechanism controlling the tissue-specific expression of the DSH1 gene.     

A mathematical model of the stress induced during avascular tumour growth

In this paper a mathematical model is developed to describe the effect of nonuniform growth on the mechanical stress experienced by cells within an avascular tumour. The constitutive law combines the stress-strain relation of linear elasticity with a growth term that is derived by analogy with thermal expansion. To accommodate the continuous nature of the growth process, the law relates the rate of change of the stress tensor to the rate of change of the strain (rather than relating the stress to the strain directly). By studying three model problems which differ in detail, certain characteristic features are identified. First, cells near the tumour boundary, where nutrient levels and cell proliferation rates are high, are under compression. By contrast, cells towards the centre of the tumour, where nutrient levels are low and cell death dominant, are under tension. The implications of these results and possible model developments are also discussed. Received: 15 November 1999 / Published online: 5 May 2000     

Myosin Va is required for P body but not stress granule formation

In the present study we demonstrate an association between mammalian myosin Va and cytoplasmic P bodies, microscopic ribonucleoprotein granules that contain components of the 5'-3' mRNA degradation machinery. Myosin Va colocalizes with several P body markers and its RNAi-mediated knockdown results in the disassembly of P bodies. Overexpression of a dominant-negative mutant of myosin Va reduced the motility of P bodies in living cells. Co-immunoprecipitation experiments demonstrate that myosin Va physically associates with eIF4E, an mRNA binding protein that localizes to P bodies. In contrast, we find that myosin Va does not play a role in stress granule formation. Stress granules are ribonucleoprotein structures that are involved in translational silencing and are spatially, functionally, and compositionally linked to P bodies. Myosin Va is found adjacent to stress granules in stressed cells but displays minimal localization within stress granules, and myosin Va knockdown has no effect on stress granule assembly or disassembly. Combined with recently published reports demonstrating a role for Drosophila and mammalian class V myosins in mRNA transport and the involvement of the yeast myosin V orthologue Myo2p in P body assembly, our results provide further evidence that the class V myosins serve an important role in the transport and turnover of mRNA.     

The MBR1 gene from Saccharomyces cerevisiae is activated by and required for growth under sub-optimal conditions

The MBR1 gene was isolated as a multicopy suppressor of the phenotype on glycerol medium of a Saccharomyces cerevisiae strain mutant for the Hap2/3/4/5 transactivator complex. In this paper, we show that Mbr1p is a limiting factor for growth on glycerol medium under the following sub-optimal culture conditions: in late growth phase, at low temperature, at high external pH or in the presence of 1,10-phenanthroline. Moreover, deletion of MBR1 prot- ects cells against stress, whilst overexpression of this gene has the opposite effect. MBR1 expression is induced in the late growth phase and is negatively controlled by the cAMP-dependent protein kinase A (PKA). Both activation of PKA or overexpression of SOK1 or SCH9– two genes isolated as multicopy suppressors of a PKA null mutant – suppress the mbr1 growth defect. Our results indicate that Mbr1p is not an essential element of any one of these pathways. Deletion of SAC1, a gene implicated in vesicular transport, in association with MBR1 deletion, causes synthetic lethality. A possible role of Mbr1p in intracellular trafficking is discussed. Received: 17 January 1997 / Accepted: 20 March 1997     

A two-component gene (NTHK1) encoding a putative ethylene-receptor homolog is both developmentally and stress regulated in tobacco

The full-length of a two-component gene NTHK1 (Nicotiana tabacum histidine kinase-l) was isolated from tobacco (N. tabacum var. Xanthi) using a previously obtained NTHK1 cDNA fragment as a probe. Sequence analysis revealed that NTHK1 shared high homology with LeETR4 from tomato and encoded an ethylene- receptor homolog. The predicted NTHK1 protein had a putative signal peptide, three transmembrane domains, a histidine kinase domain and a receiver domain. The putative autophosphorylation site at His378 and the phosphate receiver site at Asp689 were also identified. By using the in situ hybridization technique, NTHK1 mRNA was detected during flower organ development. It is also highly expressed in the processes of pollen formation and embryo development. The expression of NTHK1 in response to wounding and other stresses was investigated using competitive RT-PCR. The results demonstrated that NTHK1 was inducible upon wounding (cutting). Floating of the cut leaf pieces in 0.5× MS, with shaking, led to a relatively rapid and strong expression. This phenomenon was confirmed by the in situ hybridization results. In addition to the up-regulation by wounding, NTHK1 expression was also induced following NaCl and PEG treatment, indicating a possible role for NTHK1 in multiple stress responses. Received: 28 June 2000 / Accepted: 1 August 2000