Trichome cell growth in Arabidopsis thaliana can be derepressed by mutations in at least five genes.

Leaf trichomes in Arabidopsis are unicellular epidermal hairs with a branched morphology. They undergo successive endoreduplication rounds early during cell morphogenesis. Mutations affecting trichome nuclear DNA content, such as triptychon or glabra3, alter trichome branching. We isolated new mutants with supernumerary trichome branches, which fall into three unlinked complementation groups: KAKTUS and the novel loci, POLYCHOME and RASTAFARI. They map to chromosomes IV, II, and V, respectively. The trichomes of these mutants presented an increased DNA content, although to a variable extent. The spindly-5 mutant, which displays a constitutive gibberellin response, also produces overbranched trichomes containing more nuclear DNA. We analyzed genetic interactions using double mutants and propose that two independent pathways, defined by SPINDLY and TRIPTYCHON, act to limit trichome growth. KAKTUS and POLYCHOME might have redundant actions mediating gibberellin control via SPINDLY. The overall leaf polysomaty was not notably affected by these mutations, suggesting that they affect the control of DNA synthesis in a tissue- or cell type-specific manner. Wild-type tetraploids also produce overbranched trichomes; they displayed a shifted polysomaty in trichomes and in the whole leaf, suggesting a developmental program controlling DNA increases via the counting of endoreduplication rounds.

Expression patterns of GASA genes in Arabidopsis thaliana: the GASA4 gene is up-regulated by gibberellins in meristematic regions.

The GASA gene family previously identified in Arabidopsis belongs to a wide-spread class of genes found in mono- and dicotyledonous plants, all structurally related to the original GA-regulated GAST1 gene from tomato. They encode small peptides (97 to 112 residues) of unknown function sharing a 60 amino acid conserved C-terminal domain comprising twelve conserved cysteine residues which define a pattern not related to other known cysteine-rich motifs. Northern blot hybridization analysis revealed sequential expression of three genes during flowering, silique development and seed germination. GASA4 transcripts were detected in flower buds. GASA1 transcripts markedly accumulated in siliques, about five days after pollination, and correlated with the peak of GA biosynthesis at this stage of silique development. GASA3 transcripts accumulated at the end of the maturation stage of the silique, and transcripts were still present in dry seeds but degraded rapidly during imbibition. In addition, the GASA4 gene was again actively transcribed after germination and this expression was shown to be dependent on the presence of GAs in GA-deficient mutants. Immunoblot analysis confirmed the presence of the GASA4 gene product in flower buds, seedlings and roots. We focused on the GASA4 gene and characterized its expression. The upstream region (-890 to +128) was fused to the GUS reporter gene. GASA4/GUS expression was detected in transgenic Arabidopsis primarily in all meristematic regions, including vegetative, inflorescence and floral meristems, as well as primary and lateral root tips. In a GA-deficient background (ga1-3), GUS activity in the vegetative meristem was detected only in the presence of supplied GA. In root and flower meristems, basal GUS activity was slightly enhanced by exogenous GA. Interestingly, GA strongly inhibit GUS activity in expanding cotyledons and leaves in ga1-3 mutants supplied with exogenous GAs, as well as in the wild type. The GA-dependent meristem-specific expression pattern suggests that the GASA4 protein plays a role in dividing cells rather than in elongating cells.

GASA, a gibberellin-regulated gene family from Arabidopsis thaliana related to the tomato GAST1 gene.

A multiple gene family of at least four members, related to a GA-stimulated transcript (GAST1) from tomato, was characterized in Arabidopsis thaliana by analysing four related cDNAs, named GASA1 to GASA4. The corresponding peptides display comparable structural features: (1) a putative signal peptide of 18 to 23 residues; (2) a highly divergent hydrophilic region of about 22 amino acids; (3) a conservative 60 amino acid C-terminal domain containing 12 cysteines. This organization has also bean shown in two related peptides from tomato, GAST1 found in shoots and RSI-1 found in early lateral roots. Southern blot hybridization patterns showed single-copy genes for all four members of the GASA family. Accumulation of the various transcripts, monitored by northern blot hybridization, indicated that the various genes are expressed differentially in plant organs: Specific mRNAs were mostly detected in flower buds and immature siliques in the case of GASA1, in siliques and dry seeds in the case of GASA2 and 3, and in growing roots and flower buds in the case of GASA4. At least two of the GASA genes are activated in GA-deficient mutant ga5, as early as 4 to 8 h after spraying with 50 microM GA3. The complex patterns of expression and regulation of the various genes suggest that the related peptides are involved in a developmental regulation process in Arabidopsis.

Expression of the rpl23, rpl2 and rps19 genes in spinach chloroplasts.

The expression of the spinach rpl23, rpl2 and rps19 chloroplast genes has been studied. The rpl23 gene identified in tobacco and Marchantia, is split into two overlapping reading frames in spinach. S1 mapping has shown that initiation sites could occur upstream of each reading frames. A large transcription unit is also present covering the rpl2 and rps19 genes. The rps19 and rpl2 gene products are identified by NH2-terminal amino acid sequences. They correspond to spinach chloroplast ribosomal proteins CS-S23 and CS-L4, respectively. No product of the rpl23 gene was detected in the chloroplast 50S ribosomal subunit. This strongly suggest that a corresponding gene has been transfered into the nucleus.

Identification of an Escherichia coli S1-like protein in the spinach chloroplast ribosome.

Antibodies directed against E. coli ribosomal protein S1 were used in immunoblotting assays to search for an S1-like protein in the ribosome of spinach chloroplast. An immunological cross-reaction was reproducibly detected on the blots and inhibition experiments have demonstrated its specificity. The chloroplastic ribosomal protein which has epitopes common to antigenic determinants of the E. coli protein S1 was identified as being protein S2/S3.

In organello and in vitro phosphorylation of chloroplast ribosomal proteins.

Two chloroplast ribosomal proteins are phosphorylated in isolated chloroplast. One in the large subunit (L18) and one in the small subunit ( LS31 ). The phosphorylation is light dependent and occurs on a serine residue for both ribosomal proteins. These two proteins and other chloroplast ribosomal proteins are also phosphorylated in vitro using [gamma 32P]-ATP and a cAMP -dependent or a cAMP - independent protein kinase. The existence of a protein-kinase bound to chloroplast 70S ribosomes is also demonstrated, the enzyme is able to phosphorylate almost every chloroplast ribosomal protein.

Comparison of ribosomal proteins of chloroplast from spinach and of E. coli.

A comparison of ribosomal proteins from Escherichia coli and from chloroplasts of Spinach was made using two separate methods: electrophoretic migration and immunochemical cross-reaction between blotted E. coli ribosomal proteins and chloroplast ribosomal subunits antisera. It is shown that L2 from E. coli (E-L2) and L4 from chloroplasts (CS-L4) comigrated and that E-L2 immunologically cross-reacted with the isolated CS-L4 antibody. Co-migration was observed for three additional couples of 50S ribosomal proteins. It is also shown that at least one 30S E. coli ribosomal protein immuno-cross reacted with a 30S chloroplast antiserum and that three couples of 30S ribosomal proteins comigrated.

Site of synthesis of spinach chloroplast ribosomal proteins and formation of incomplete ribosomal particles in isolated chloroplasts.

Chloroplast ribosomal proteins from spinach have been prepared in the presence of a protease inhibitor and some modifications have been introduced to the previous characterization of the 50S subunits (Mache et al., MGG, 177, 333, 1980): 33 ribosomal proteins are detected instead of 34. No change has been observed for the 30S subunits.Using a light-driven system of protein synthesis it is shown that up to ten ribosomal proteins of the 30S and eight proteins of the 50S subunits are made in the chloroplast.Newly synthesized ribosomal subunits have been analysed on CsCl gradients after sedimentation at equilibrium, allowing the separation of fully assembled subunits from incomplete ribosomal particles. Most of the newly made 50S subunits are fully assembled (ρ=1.634). A small amount of incomplete 50S particles (ρ=1.686) is detectable. Newly made 30S subunits (ρ=1.598) and incomplete 30S particles (ρ=1.691) are also observed. The ribosomal proteins of the incomplete 30S have been determined. They contain eight or nine of the 30S-proteins, seven of which are synthesized within the chloroplast. It is suggested that incomplete ribosomal particles resulted from a step in the assembly of ribosomal subunits.