Michael Schläppi

Competence of Immature Maize Embryos for Agrobacterium-Mediated Gene Transfer.

Agrobacterium-mediated transfer of viral sequences to plant cells (agroinfection) was applied to study the susceptibility of immature maize embryos to the pathogen. The shoot apical meristem of immature embryos 10 to 20 days after pollination from four different maize genotypes was investigated for competence for agroinfection. There was a direct correlation between different morphological stages of the unwounded immature embryos and their competence for agroinfection. Agroinfection frequency was highest in the embryogenic line A188. All developmental stages tested showed Agrobacterium virulence gene-inducing activity, whereas bacteriocidal substances were produced at stages of the immature embryos competent for agroinfection. The results suggested that Agrobacterium may require differentiated tissue in the maize shoot apical meristem before wounding for successful T-DNA transfer. This requirement for the young maize embryo has implications for the possible use of Agrobacterium for maize transformation.

Epigenetic regulation of the maize Spm transposable element : novel activation of a methylated promoter by TnpA

Abstract Spm is epigenetically inactivated by C-methylation near its transcription start site. We have investigated the interaction between TnpA, an autoregulatory protein that can reactivate a silent Spm , and the promoter of the element. The promoter undergoes rapid de novo methylation and inactivation in stably transformed plants, but only if it includes a GC-rich sequence downstream of the promoter. TnpA activates the inactive, methylated promoter and leads to reduced methylation. By contrast, TnpA represses the active, unmethylated Spm promoter. Only the internal DNA-binding and dimerization domains of the protein are required for repression, while activation requires an additional C-terminal sequence. TnpA is therefore a unique regulatory protein with a conventional transcriptional repressor activity and a novel ability to activate a methylated, inactive promoter.

FRIGIDA LIKE 2 Is a Functional Allele in Landsberg erecta and Compensates for a Nonsense Allele of FRIGIDA LIKE 1

The Landsberg erecta (Ler) accession of Arabidopsis (Arabidopsis thaliana) has a weak allele of the floral inhibitor FLOWERING LOCUS C (FLC). FLC-Ler is weakly up-regulated by the active San Feliu-2 (Sf2) allele of FRIGIDA (FRI-Sf2), resulting in a moderately late-flowering phenotype. By contrast, the Columbia (Col) allele of FLC is strongly up-regulated by FRI-Sf2, resulting in a very late-flowering phenotype. In Col, the FRI-related gene FRI LIKE 1 (FRL1) is required for FRI-mediated up-regulation of FLC. It is shown here that in Ler, the FRL1-related gene FRI LIKE 2 (FRL2), but not FRL1, is required for FRI-mediated up-regulation of FLC. FRL1-Ler is shown to be a nonsense allele of FRL1 due to a naturally occurring premature stop codon in the middle of the conceptual protein sequence, suggesting that FRL1-Ler is nonfunctional. Compared to FRL2-Col, FRL2-Ler has two amino acid changes in the conceptual protein sequence. Plants homozygous for FRI-Sf2, FLC-Ler, FRL1-Ler, and FRL2-Col have no detectable FLC expression, resulting in an extremely early flowering phenotype. Transformation of a genomic fragment of FRL2-Ler, but not of FRL2-Col, into a recombinant inbred line derived from these plants restores both FRI-mediated up-regulation of FLC expression and a late-flowering phenotype, indicating that FRL2-Ler is the functional allele of FRL2. Taken together, these results suggest that in the two different Arabidopsis accessions Col and Ler, either FRL1 or FRL2, but not both, is functional and required for FRI-mediated up-regulation of FLC.

Cold responsive EARLI1 type HyPRPs improve freezing survival of yeast cells and form higher order complexes in plants.

Plants have large families of proteins sharing a conserved eight-cysteine-motif (8CM) domain. The biological functions of these proteins are largely unknown. EARLI1 is a cold responsive Arabidopsis gene that encodes a hybrid proline-rich protein (HyPRP) with a three-domain architecture: a putative signal peptide at the N-terminus, a proline-rich domain (PRD) in the middle, and an 8CM domain at the C-terminus. We report here that yeast cells expressing different EARLI1 genes had significantly higher rates of freezing survival than empty-vector transformed controls. Arabidopsis plants with knocked down EARLI1 genes had an increased tendency for freezing-induced cellular damage. EARLI1-GFP fluorescence in transgenic plants and immunoblot analyses using protoplasts suggested cell wall localization for EARLI1 proteins. Immunoblot analyses showed that EARLI1 proteins form higher order complexes in plants, and that the PRD is a soluble and the 8CM an insoluble protein domain. We propose that EARLI1 proteins have a bimodular architecture in which the PRD may interact with the cell wall and the 8CM domain with the plasma membrane to protect the cells during freezing stress.

A Gene Expression Screen Identifies EARLI1 as a Novel Vernalization-Responsive Gene in Arabidopsis Thaliana

Vernalization promotes early flowering in late ecotypes of Arabidopsis thaliana. The mechanisms of vernalization are poorly understood. A subtractive hybridization approach was used to isolate vernalization-responsive genes from a late-flowering ecotype of Arabidopsis thaliana based on the premise that transcript levels of such genes would increase with cold treatment and remain high even after removal of the vernalization stimulus. EARLI1 is the first Arabidopsis gene shown to be stably activated by vernalization. The abundance of its RNA is progressively elevated by vernalization and remains high for at least 20 days at room temperature. The basal level of EARLI1 RNA is higher in early-flowering ecotypes, but is increased also after vernalization. Vernalization and subsequent growth in long-day photoperiods have an additive or synergistic effect on EARLI1 activation. EARLI1 RNA levels are also transiently induced by brief exposures to cold, but not to abscisic acid. EARLI1 is thus a novel vernalization-responsive gene in Arabidopsis thaliana that can be used to investigate vernalization-specific transcriptional regulation.

The HyPRP gene EARLI1 has an auxiliary role for germinability and early seedling development under low temperature and salt stress conditions in Arabidopsis thaliana

The effect of the hybrid proline-rich protein (HyPRP) gene EARLI1 on the rate of germination (germinability) of Arabidopsis seeds and seedling growth under low temperature and salt stress conditions was investigated. EARLI1 was induced during germination in embryonic tissues, and was strongly expressed in certain parts of young seedlings. Comparisons of control, overexpressing (OX), and knockout (KO) lines indicated that higher than wild type levels of EARLI1 improved germinability, root elongation, and reduction of sodium accumulation in leaves under salt stress, as well as germinability under low-temperature stress. Abscisic acid (ABA) contents were relatively low after prolonged salt stress, suggesting that EARLI1 has an ABA-independent effect on germinability under these conditions. Overexpression of EARLI1 during germination enhanced the sensitivity of seeds to exogenously applied ABA, suggesting that EARLI1 has an ABA-dependent negative effect on seed germinability under high ABA stress conditions. Well-known stress response marker genes such as COR15a, KIN1, P5SC1, and RD29 were unaffected whereas P5SC2, RD22, or RAB18 were only slightly affected in OX and KO plants. The pleiotropic effects of EARLI1 during stress and an absence of strong regulatory effects on stress marker genes suggest that this HyPRP gene has an auxiliary role for various stress protection responses in Arabidopsis.

Cold-inducible expression of AZI1 and its function in improvement of freezing tolerance of Arabidopsis thaliana and Saccharomyces cerevisiae

AZI1 (AZELAIC ACID INDUCED 1) of Arabidopsis thaliana could be induced by azelaic acid and was involved in priming of systemic plant immunity. In the present work, expression of AZI1 in response to low temperature was investigated via RNA gel blot analysis. AZI1 could be induced slowly by cold stress and more than 6h treatment at 4°C was required to detect an increase in mRNA abundance. However, the high expression state could not be maintained stably and would decline to basal level when the plants were transferred to room temperature. In order to clarify the function of AZI1 in resistance to abiotic stresses, overexpressing, RNA interference and T-DNA knockout lines of this gene were used in electrolyte leakage assays. Overexpression of AZI1 resulted in reduced electrolyte leakage during freezing damage. In contrast, AZI1 knockdown and knockout lines showed increased tendencies in cellular damage after freezing treatment. To further validate the potential resistance of AZI1 to low-temperature stress, Saccharomyces cerevisiae cells were transformed with pESC-AZI1 in which AZI1 was under the control of GAL1 promoter. Compared to yeast cells containing empty pESC-URA, the survival rate of yeast cells harboring AZI1 increased obviously after freezing treatment. All these results suggested that AZI1 might be multifunctional and associated with cold tolerance of Arabidopsis.

RNA LEVELS AND ACTIVITY OF FLOWERING LOCUS C ARE MODIFIED IN MIXED GENETIC BACKGROUNDS OF ARABIDOPSIS THALIANA

Flowering time and FLOWERING LOCUS C (FLC) RNA levels were analyzed in different accessions of Arabidopsis thaliana and in mixed genetic backgrounds resulting from crosses between accessions. Dominant alleles of FRIGIDA (FRI) promote accumulation of FLC RNA, which in turn promotes late flowering. Although the coding regions of sequenced FLC alleles are identical, some accessions have genetically weak alleles that do not promote late flowering in the presence of FRI. In this study, a new weak allele of FLC with open reading frame identity to previously sequenced alleles was isolated from a Niederzenz (Nd) accession. The FLC‐Nd allele accumulated less RNA in the presence of FRI than did the strong Columbia (Col) allele. The weak FLC‐Nd allele was semidominant in the mixed Nd/Col genetic background containing FRI, and a linear correlation between the level of FLC RNA and lateness of flowering was observed. However, late‐flowering transgressions with elevated levels of FLC RNA in the absence of FRI were also obtained from crosses between early‐flowering accessions Col and Nd. Moreover, compared to Nd, the weak Landsberg erecta (Ler) allele of FLC was recessive and not semidominant in the mixed Ler/Col genetic background. However, very early‐flowering transgressions lacking detectable FLC RNA were also obtained from crosses between FRI containing Col and Ler. The results indicate that modifier genes other than FRI influence the level and genetic activity of FLC RNA in different genetic backgrounds resulting from crosses between naturally occurring accessions of A. thaliana.

A highly sensitive plant hybrid protein assay system based on the Spm promoter and TnpA protein for detection and analysis of transcription activation domains

TnpA is a multifunctional DNA binding protein encoded by the maize Suppressor-mutator (Spm) transposable element. TnpA is required for transposition and is a repressor of the unmethylated Spm promoter. While analyzing protein domains using a yeast GAL4-based hybrid system in transiently transformed tobacco cells, we found that TnpA represses the >10-fold transcriptional activation observed when the GAL4 DNA-binding domain is used alone. By contrast, compared to the backgroundless TnpA DNA-binding domain alone, 33-to 45-fold activation of the Spm promoter was observed when the VP16 activation domain was fused to it. TnpA-binding sites, but no TATA box, were required for transcription activation. Among the TnpA deletion derivatives tested, those retaining the coding sequences for the DNA-binding and protein dimerization domains gave the highest level of transcription activation when fused with the VP16 activation domain. The TnpA gene and TnpA-binding sites in the short Spm promoter therefore provide a novel, highly sensitive single-hybrid system for identifying and studying plant transcription activation domains in plant cells.

Influence of EARLI1-like genes on flowering time and lignin synthesis of Arabidopsis thaliana

EARLI1 encodes a 14.7 kDa protein in the cell wall, is a member of the PRP (proline-rich protein) family and has multiple functions, including resistance to low temperature and fungal infection. RNA gel blot analyses in the present work indicated that expression of EARLI1-like genes, EARLI1, At4G12470 and At4G12490, was down-regulated in Col-FRI-Sf2 RNAi plants derived from transformation with Agrobacterium strain ABI, which contains a construct encoding a double-strand RNA targeting 8CM of EARLI1. Phenotype analyses revealed that Col-FRI-Sf2 RNAi plants of EARLI1 flowered earlier than Col-FRI-Sf2 wild-type plants. The average bolting time of Col-FRI-Sf2 and Col-FRI-Sf2 RNAi plants was 39.7 and 19.4 days, respectively, under a long-day photoperiod. In addition, there were significant differences in main stem length, internode number and rosette leaf number between Col-FRI-Sf2 and Col-FRI-Sf2 RNAi plants. RT-PCR showed that EARLI1-like genes might delay flowering time through the autonomous and long-day photoperiod pathways by maintaining the abundance of FLC transcripts. In Col-FRI-Sf2 RNAi plants, transcription of FLC was repressed, while expression of SOC1 and FT was activated. Microscopy observations showed that EARLI1-like genes were also associated with morphogenesis of leaf cells in Arabidopsis. Using histochemical staining, EARLI1-like genes were found to be involved in regulation of lignin synthesis in inflorescence stems, and Col-FRI-Sf2 and Col-FRI-Sf2 RNAi plants had 9.67% and 8.76% dry weight lignin, respectively. Expression analysis revealed that cinnamoyl-CoA reductase, a key enzyme in lignin synthesis, was influenced by EARLI1-like genes. These data all suggest that EARLI1-like genes could control the flowering process and lignin synthesis in Arabidopsis.