Nitrate or NaCl regulates floral induction in Arabidopsis thaliana

Flowering, the transition from the vegetative to reproductive phase in plants, is regulated by both endogenous and environmental signals. Exposure to an extended period of stress (such as low nitrate or NaCl) can also promote flowering in many species, but little is known about how these forms of stress regulate floral induction. In this study, we found that stress induced by low concentrations of nitrate or NaCl activated the biosynthesis of gibberellin (GA) as evidenced by increased expression of the GA biosynthetic enzyme GA1. Expression of CO and SOC1 were also enhanced, leading to an acceleration of flowering. The effects of nitrate and NaCl on the photoperiod pathway were distinct, however. Two genes related to the photoperiod pathway, CCA1 and LHY, were repressed only under low NaCl treatment, while expression was unaltered by nitrate. Therefore, we suggest that the biosynthesis of gibberellin (GA) may play an important role in integrating signals induced by exogenous stress to regulate flowering in Arabidopsis.     

Pumpkin phloem lectin genes are specifically expressed in companion cells.

Pumpkin phloem exudate contains two abundant phloem proteins: PP1 is a 96-kD protein that forms polymeric filaments in vivo, and PP2 is a 48-kD dimeric lectin. Polyclonal antibodies raised against pumpkin phloem exudate were used to isolate several cDNAs corresponding to PP1 and PP2. RNA gel blot analysis indicated that PP1 is encoded by an mRNA of approximately 2500 nucleotides, whereas PP2 subunits are encoded by an mRNA of 1000 nucleotides. Sequence analysis of PP2 cDNAs revealed a 654-bp open reading frame encoding a 218-amino acid polypeptide; this polypeptide had the carbohydrate binding characteristics of a PP2 subunit. The PP2 mRNA was localized within the phloem of pumpkin hypocotyl cross-sections based on in situ hybridization of a digoxigenin-labeled antisense probe. PP2 mRNA was found within the companion cells in both the bicollateral vascular bundles and the extrafascicular phloem network.     

Arabidopsis NPCC6/NaKR1 is a phloem mobile metal binding protein necessary for phloem function and root meristem maintenance

SODIUM POTASSIUM ROOT DEFECTIVE1 (NaKR1; previously called NPCC6) encodes a soluble metal binding protein that is specifically expressed in companion cells of the phloem. The nakr1-1 mutant phenotype includes high Na(+), K(+), Rb(+), and starch accumulation in leaves, short roots, late flowering, and decreased long-distance transport of sucrose. Using traditional and DNA microarray-based deletion mapping, a 7-bp deletion was found in an exon of NaKR1 that introduced a premature stop codon. The mutant phenotypes were complemented by transformation with the native gene or NaKR1-GFP (green fluorescent protein) and NaKR1-β-glucuronidase fusions driven by the native promoter. NAKR1-GFP was mobile in the phloem; it moved from companion cells into sieve elements and into a previously undiscovered symplasmic domain in the root meristem. Grafting experiments revealed that the high Na(+) accumulation was due mainly to loss of NaKR1 function in the leaves. This supports a role for the phloem in recirculating Na(+) to the roots to limit Na(+) accumulation in leaves. The onset of root phenotypes coincided with NaKR1 expression after germination. The nakr1-1 short root phenotype was due primarily to a decreased cell division rate in the root meristem, indicating a role in root meristem maintenance for NaKR1 expression in the phloem.     

Expression of murine leukemia virus envelope protein is sufficient for the induction of apoptosis

The generation of cytopathic effects by murine leukemia viruses (MLVs) in different cell types correlates with the ability of the virus to induce thymic lymphoma. We showed that the induction of apoptosis in mink epithelial cells by mink cell focus-forming (MCF) MLV infection results in the accumulation of high levels of both unintegrated viral DNA and the envelope precursor polyprotein (gPr80^env). Comparisons of envelope protein expression levels of plasmid clones of the env gene of the MCF13 and noncytopathic NZB-9 MLV strains demonstrated that the accumulation of MCF13 gPr80^env results in endoplasmic reticulum stress and is sufficient for the induction of apoptosis.     

FPA, a gene involved in floral induction in Arabidopsis, encodes a protein containing RNA-recognition motifs

FPA is a gene that regulates flowering time in Arabidopsis via a pathway that is independent of daylength (the autonomous pathway). Mutations in FPA result in extremely delayed flowering. FPA was identified by means of positional cloning. The predicted FPA protein contains three RNA recognition motifs in the N-terminal region. FPA is expressed most strongly in developing tissues, similar to the expression of FCA and LUMINIDEPENDENS, two components of the autonomous pathway previously identified. Overexpression of FPA in Arabidopsis causes early flowering in noninductive short days and creates plants that exhibit a more day-neutral flowering behavior.     

Hexose transporter CsSWEET7a in cucumber mediates phloem unloading in companion cells for fruit development

In the fleshy fruit of cucumbers (Cucumis sativus L.), the phloem flow is unloaded via an apoplasmic pathway, which requires protein carriers to export sugars derived from stachyose and raffinose into the apoplasm. However, transporter(s) involved in this process remain unidentified. Here, we report that a hexose transporter, CsSWEET7a (Sugar Will Eventually be Exported Transporter 7a), was highly expressed in cucumber sink tissues and localized to the plasma membrane in companion cells of the phloem. Its expression level increased gradually during fruit development. Down-regulation of CsSWEET7a by RNA interference (RNAi) resulted in smaller fruit size along with reduced soluble sugar levels and reduced allocation of ¹⁴C-labelled carbon to sink tissues. CsSWEET7a overexpression lines showed an opposite phenotype. Interestingly, genes encoding alkaline α-galactosidase (AGA) and sucrose synthase (SUS) were also differentially regulated in CsSWEET7a transgenic lines. Immunohistochemical analysis demonstrated that CsAGA2 co-localized with CsSWEET7a in companion cells, indicating cooperation between AGA and CsSWEET7a in fruit phloem unloading. Our findings indicated that CsSWEET7a is involved in sugar phloem unloading in cucumber fruit by removing hexoses from companion cells to the apoplasmic space to stimulate the raffinose family of oligosaccharides (RFOs) metabolism so that additional sugars can be unloaded to promote fruit growth. This study also provides a possible avenue towards improving fruit production in cucumber.

Transporter CsSWEET7a removes hexose from companion cells to the apoplasmic space to stimulate fruit phloem unloading so that additional sugars can be unloaded to promote fruit growth.     

Expression of phosphophoryn is sufficient for the induction of matrix mineralization by mammalian cells

Mineralized tissues such as dentin and bone assemble extracellular matrices uniquely rich in a variety of acidic phosphoproteins. Although these proteins are presumed to play a role in the process of biomineralization, key questions regarding the nature of their contributions remain unanswered. First, it is not known whether highly phosphorylated proteins alone can induce matrix mineralization, or whether this activity requires the involvement of other bone/dentin non-collagenous proteins. Second, it remains to be established whether the protein kinases that phosphorylate these acidic proteins are unique to cells responsible for producing mineralized tissues. To begin to address these questions, we consider the case of phosphophoryn (PP), due to its high content of phosphate, high affinity for Ca(2+), and its potential role in hydroxyapatite nucleation. We have created a model system of biomineralization in a cellular environment by expressing PP in NIH3T3 fibroblasts (which do not produce a mineralized matrix); as a positive control, PP was expressed in MC3T3-E1 osteoblastic cells, which normally mineralize their matrices. We show that expression of PP in NIH3T3 cells is sufficient for the induction of matrix mineralization. In addition, assessment of the phosphorylation status of PP in these cells reveals that the transfected NIH3T3 cells are able to phosphorylate PP. We suggest that the phosphorylation of PP is essential for mineral formation. The principle goal of this study is to enrich the current knowledge of mineralized tissue phosphorylation events by analyzing them in the context of a complete cellular environment.     

Structure and development of P-protein in phloem parenchyma and companion cells of legumes.

Phloem parenchyma and companion cells from four species of legumes, Phaseolus vulgaris L., Melilotus alba Desr., Desmodium canadense, L. and Dolichos lablab L., were examined electron microscopically to estabish the presence, structure and development of P-protein. P-protein components consisting of granular, fibrillar, tubular or crystalline structures were found in parenchyma cells of all species and in companion cells of M. alba. The earliest stages of P-protein formation were closely associated with dictyosome cisternae, dictyosome vesicles and/or spiny vesicles. After their formation, the P-protein bodies were frequently transformed into one or more structurally different components. Although these components appeared to be develop-mentally related, their specific associations and transformations differed in each species examined.     

The N-terminal ATPase AT-hook-containing region of the Arabidopsis chromatin-remodeling protein SPLAYED is sufficient for biological activity

The SNF2-like chromatin-remodeling ATPase SPLAYED (SYD) was identified as a co-activator of floral homeotic gene expression in Arabidopsis. SYD is also required for meristem maintenance and regulates flowering under a non-inductive photoperiod. SNF2 ATPases are structurally and functionally conserved from yeast to humans. In addition to the conserved protein features, SYD has a large unique C-terminal domain. We show here that SYD is present as two forms in the nucleus, full-length and truncated, with the latter apparently lacking the C-terminal domain. The ratio of the two forms of endogenous SYD differs in juvenile and in adult tissues. Furthermore, an SYD variant lacking the C-terminal domain (SYDDeltaC) rescues the syd null mutant, indicating that the N-terminal ATPase AT-hook-containing region of SYD is sufficient for biological activity. Plants expressing SYDDeltaC show molecular and morphological phenotypes opposite to those of the null mutant, suggesting that the construct results in increased activity. This increased activity is at least in part due to elevated SYD protein levels in these lines. We propose that the C-terminal domain may control SYD accumulation and/or specific activity in the context of the full-length protein. The presence of the C-terminal domain in rice SYD suggests that its role is probably conserved in the two classes of flowering plants.     

Arabidopsis nitrate transporter NRT1.9 is important in phloem nitrate transport

This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport.     

Brahma is required for proper expression of the floral repressor FLC in Arabidopsis

Background

BRAHMA (BRM) is a member of a family of ATPases of the SWI/SNF chromatin remodeling complexes from Arabidopsis. BRM has been previously shown to be crucial for vegetative and reproductive development.

Methodology/Principal Findings

Here we carry out a detailed analysis of the flowering phenotype of brm mutant plants which reveals that, in addition to repressing the flowering promoting genes CONSTANS (CO), FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1), BRM also represses expression of the general flowering repressor FLOWERING LOCUS C (FLC). Thus, in brm mutant plants FLC expression is elevated, and FLC chromatin exhibits increased levels of histone H3 lysine 4 tri-methylation and decreased levels of H3 lysine 27 tri-methylation, indicating that BRM imposes a repressive chromatin configuration at the FLC locus. However, brm mutants display a normal vernalization response, indicating that BRM is not involved in vernalization-mediated FLC repression. Analysis of double mutants suggests that BRM is partially redundant with the autonomous pathway. Analysis of genetic interactions between BRM and the histone H2A.Z deposition machinery demonstrates that brm mutations overcome a requirement of H2A.Z for FLC activation suggesting that in the absence of BRM, a constitutively open chromatin conformation renders H2A.Z dispensable.

Conclusions/Significance

BRM is critical for phase transition in Arabidopsis. Thus, BRM represses expression of the flowering promoting genes CO, FT and SOC1 and of the flowering repressor FLC. Our results indicate that BRM controls expression of FLC by creating a repressive chromatin configuration of the locus.     

The nature of floral signals in Arabidopsis. II. Roles for FLOWERING LOCUS T (FT) and gibberellin

Signals produced in leaves are transported to the shoot apex where they cause flowering. Protein of the gene FLOWERING LOCUS T (FT) is probably a long day (LD) signal in Arabidopsis. In the companion paper, rapid LD increases in FT expression associated with flowering driven photosynthetically in red light were documented. In a far red (FR)-rich LD, along with FT there was a potential role for gibberellin (GA). Here, with the GA biosynthesis dwarf mutant ga1-3, GA(4)-treated plants flowered after 26 d in short days (SD) but untreated plants were still vegetative after 6 months. Not only was FT expression low in SD but applied GA bypassed some of the block to flowering in ft-1. On transfer to LD, ga1-3 only flowered when treated simultaneously with GA, and FT expression increased rapidly (<19.5 h) and dramatically (15-fold). In contrast, in the wild type in LD there was little requirement for GA for FT increase and flowering so its endogenous GA content was near to saturating. Despite this permissive role for endogenous GA in Columbia, RNA interference (RNAi) silencing of the GA biosynthesis gene, GA 20-OXIDASE2, revealed an additional, direct role for GA in LD. Flowering took twice as long after silencing the LD-regulated gene, GA 20-OXIDASE2. Such independent LD input by FT and GA reflects their non-sympatric expression (FT in the leaf blade and GA 20-OXIDASE2 in the petiole). Overall, FT acts as the main LD floral signal in Columbia and GA acts on flowering both via and independently of FT.     

Glutamate dehydrogenase of tobacco is mainly induced in the cytosol of phloem companion cells when ammonia is provided either externally or released during photorespiration

Glutamate (Glu) dehydrogenase (GDH) catalyses the reversible amination of 2-oxoglutarate for the synthesis of Glu using ammonium as a substrate. This enzyme preferentially occurs in the mitochondria of companion cells of a number of plant species grown on nitrate as the sole nitrogen source. For a better understanding of the controversial role of GDH either in ammonium assimilation or in the supply of 2-oxoglutarate (F. Dubois, T. Terce-Laforgue, M.B. Gonzalez-Moro, M.B. Estavillo, R. Sangwan, A. Gallais, B. Hirel [2003] Plant Physiol Biochem 41: 565-576), we studied the localization of GDH in untransformed tobacco (Nicotiana tabacum) plants grown either on low nitrate or on ammonium and in ferredoxin-dependent Glu synthase antisense plants. Production of GDH and its activity were strongly induced when plants were grown on ammonium as the sole nitrogen source. The induction mainly occurred in highly vascularized organs such as stems and midribs and was likely to be due to accumulation of phloem-translocated ammonium in the sap. GDH induction occurred when ammonia was applied externally to untransformed control plants or resulted from photorespiratory activity in transgenic plants down-regulated for ferredoxin-dependent Glu synthase. GDH was increased in the mitochondria and appeared in the cytosol of companion cells. Taken together, our results suggest that the enzyme plays a dual role in companion cells, either in the mitochondria when mineral nitrogen availability is low or in the cytosol when ammonium concentration increases above a certain threshold.     

HUA2 is required for the expression of floral repressors in Arabidopsis thaliana

The HUA2 gene acts as a repressor of floral transition. Lesions in hua2 were identified through a study of natural variation and through two mutant screens. An allele of HUA2 from Landsberg erecta (Ler) contains a premature stop codon and acts as an enhancer of early flowering 4 (elf4) mutants. hua2 single mutants, in the absence of the elf4 lesion, flower earlier than wild type under short days. hua2 mutations partially suppress late flowering in FRIGIDA (FRI )-containing lines, autonomous pathway mutants, and a photoperiod pathway mutant. hua2 mutations suppress late flowering by reducing the expression of several MADS genes that act as floral repressors including FLOWERING LOCUS C (FLC ) and FLOWERING LOCUS M (FLM ).     

The Arabidopsis floral homeotic gene PISTILLATA is regulated by discrete cis-elements responsive to induction and maintenance signals

PISTILLATA is a B-class floral organ identity gene required for the normal development of petals and stamens in Arabidopsis. PISTILLATA expression is induced in the stage 3 flowers (early expression) and is maintained until anthesis (late expression). To explore in more detail the developmentally regulated gene expression of PISTILLATA, we have analyzed the PISTILLATA promoter using uidA (beta)-glucuronidase gene) fusion constructs (PI::GUS) in transgenic Arabidopsis. Promoter deletion analyses suggest that early PISTILLATA expression is mediated by the distal region and that late expression is mediated by the proximal region. Based on the PI::GUS expression patterns in the loss- and gain-of-function alleles of meristem or organ identity genes, we have shown that LEAFY and UNUSUAL FLORAL ORGANS induce PISTILLATA expression in a flower-independent manner via a distal promoter, and that PISTILLATA and APETALA3 maintain PISTILLATA expression (autoregulation) in the later stages of flower development via a proximal promoter. In addition, we have demonstrated that de novo protein synthesis is required for the PISTILLATA autoregulatory circuit.