Overexpression of KNAT1 in lettuce shifts leaf determinate growth to a shoot-like indeterminate growth associated with an accumulation of isopentenyl-type cytokinins.

Leaves are specialized organs characterized by defined developmental destiny and determinate growth. The overexpression of Knotted1-like homeobox genes in different species has been shown to alter leaf shape and development, but a definite role for this class of genes remains to be established. Transgenics that overexpress Knotted1-like genes present some traits that are characteristic of altered cytokinin physiology. Here we show that lettuce (Lactuca sativa) leaves that overexpress KNAT1, an Arabidopsis kn1-like gene, acquire characteristics of indeterminate growth typical of the shoot and that this cell fate change is associated with the accumulation of specific types of cytokinins. The possibility that the phenotypic effects of KNAT1 overexpression may arise primarily from the modulation of local ratios of different cytokinins is discussed.

Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development.

Auxin plays a key role in lateral root formation, but the signaling pathway for this process is poorly understood. We show here that NAC1, a new member of the NAC family, is induced by auxin and mediates auxin signaling to promote lateral root development. NAC1 is a transcription activator consisting of an N-terminal conserved NAC-domain that binds to DNA and a C-terminal activation domain. This factor activates the expression of two downstream auxin-responsive genes, DBP and AIR3. Transgenic plants expressing sense or antisense NAC1 cDNA show an increase or reduction of lateral roots, respectively. Finally, TIR1-induced lateral root development is blocked by expression of antisense NAC1 cDNA, and NAC1 overexpression can restore lateral root formation in the auxin-response mutant tir1, indicating that NAC1 acts downstream of TIR1.

Isolation and molecular characterisation of the gene encoding the cytoplasmic ribosomal protein S28 in Prunus persica [L.]] Batsch.

RT-PCR was performed on peach (Prunus persica [L.] Batsch) RNA to isolate cDNAs corresponding to transcripts which are differentially expressed in leaves borne on basal and apical shoots. A gene was identified which was more highly expressed in the leaves of basal shoots, and codes for the cytoplasmic protein S28 present in the small ribosomal subunit. The 5' leader regions of RPS28 mRNAs were found to harbour 8-11 pyrimidine tracts, which suggested similarities to regulatory stretches that control the translation of mRNAs for ribosomal proteins in animals. The peach S28 is encoded by two intron-containing genes, which are both transcribed in mitotically active tissues such as developing leaves and roots. In situ hybridisation to shoot vegetative apices and the measurement of nucleus/nucleolus ratios indicated that RPS28 expression was confined to areas undergoing active cell division. The mature RPS28 mRNA was detected as a single species in actively dividing tissues such as apical tips, developing leaves, vegetative buds, stamens, developing fruits and roots. In contrast, accumulation of a precursor RNA, in the presence of the mature product, was found in fully expanded leaves and subtending stems, while only the precursor species was detected in several late-stage tissues. This phenomenon suggested that expression of the mature RNA is controlled at the level of splicing and turnover of the precursor RNA. This is similar to the mode of regulation of ribosomal protein genes in animals.

MsJ1, an alfalfa DnaJ-like gene, is tissue-specific and transcriptionally regulated during cell cycle.

DnaJ-like proteins are molecular chaperones that regulate Hsp70 ATPase activity both in protein folding, assembly and disassembly of protein complexes. Here we report the isolation of MsJ1, an alfalfa gene encoding a protein homologous to cytosolic DnaJ-like proteins. MsJ1 was induced under heat-shock treatment in both leaves and stems of adult plants. In the absence of heat shock MsJ1 expression was tissue-specific with the highest levels of mRNA in roots and in embryonal structures. High levels of transcript were also detected in cotyledons where active degradation of storage protein occurs. In synchronized alfalfa suspension-cultured cells the MsJ1 transcript was actively expressed and showed a phase-specific modulation during cell cycle with a 2-fold induction in G2/M. These findings suggest that DnaJ-like proteins play an active role in regulating normal cellular events like protein degradation, morphogenesis and cell cycle progression.

Agrobacterium rhizogenes rol genes induce productivity-related phenotypical modifications in "creeping-rooted" alfalfa types.

Agrobacterium rhizogenes rol genes were transferred individually or in combination into the forage legume Medicago sativa L. (alfalfa). Kanamycin resistant, neomycin phosphotransferase II positive plants showed the presence of the rol inserts in their genome. Phenotypical evaluation of transgenic populations indicated significant morphological alterations of the root system, stem number per plant and plant structure. A possible utilization of these transgenics in breeding programs of the so-called "creeping-rooted" alfalfa strains is discussed.

Expression in different populations of cells of the root meristem is controlled by different domains of the rolB promoter.

Selective gene expression in different populations of cells of the root apex of transgenic tobacco could be evidenced by means of GUS constructs with deletions of the rolB promoter and fusions with the CaMV 35S minimal promoter. Five regulatory regions have been broadly identified in the rolB 5' non-coding region. The presence of all five domains (A to E) directs gene expression in the root cap, in the protoderm and in the different tissues within the root meristematic region: the dermatocalyptrogen, the cortex and the vascular cylinder. Deletion of domain A (-623 to -471) selectively suppresses expression in non-meristematic cells, i.e. the root cap and the protoderm. Deletion of either domain B (-341 to -306) or E (80 bp around the TATA box) causes loss of expression in all cells of the root apex: constructs C + D + E, B + C + D, B + C are inactive. Domain D (70 bp around the CAAT box) is necessary for gene expression in the dermatogen and in meristematic cells of the cortex but not in the innermost meristematic layer: construct B + C + E is active only in vascular meristematic cells. Domain C (-216 to -158) seems to have a double regulatory role as construct B + E is no longer expressed in meristematic cells of the vascular cylinder but is very active in the protoderm. Constructs allowing gene expression in meristematic cells are also inducible by auxin in leaf protoplasts, while activation of the regulatory elements necessary for gene expression in the non-meristematic cells of the root apex do not seem to depend upon the hormone. The connection between auxin induction and meristematic expression is discussed.

Genetic transformation in the grain legume Cicer arietinum L. (chickpea).

In the grain legume Cicer arietinum L. (chickpea), the seed-derived embryo axes deprived of the apical meristem were able to regenerate adventitious shoots on Murashige and Skoog (1962) medium supplemented with kinetin. This protocol was suitable for Agrobacterium-mediated gene transfer by the co-cultivation technique. Chickpea transgenic plants showed neomycin phosphotransferase II and ß-glucuronidase activities and the presence in their genome of integrated bacterial DNA.