Mutagenesis of plants overexpressing CONSTANS demonstrates novel interactions among Arabidopsis flowering-time genes.

CONSTANS (CO) promotes flowering of Arabidopsis in response to long photoperiods. Transgenic plants carrying CO fused with the cauliflower mosaic virus 35S promoter (35S::CO) flowered earlier than did the wild type and were almost completely insensitive to length of day. Genes required for CO to promote flowering were identified by screening for mutations that suppress the effect of 35S::CO. Four mutations were identified that partially suppressed the early-flowering phenotype caused by 35S::CO. One of these mutations, suppressor of overexpression of CO 1 (soc1), defines a new locus, demonstrating that the mutagenesis approach is effective in identifying novel flowering-time mutations. The other three suppressor mutations are allelic with previously described mutations that cause late flowering. Two of them are alleles of ft, indicating that FT is required for CO to promote early flowering and most likely acts after CO in the hierarchy of flowering-time genes. The fourth suppressor mutation is an allele of fwa, and fwa soc1 35S::CO plants flowered at approximately the same time as co mutants, suggesting that a combination of fwa and soc1 abolishes the promotion of flowering by CO. Besides delaying flowering, fwa acted synergistically with 35S::CO to repress floral development after bolting. The latter phenotype was not shown by any of the progenitors and was most probably caused by a reduction in the function of LEAFY. These genetic interactions suggest models for how CO, FWA, FT, and SOC1 interact during the transition to flowering.

The Chlamydomonas reinhardtii MoCo carrier protein is multimeric and stabilizes molybdopterin cofactor in a molybdate charged form.

In Chlamydomonas reinhardtii, molybdopterin cofactor (MoCo) able to reconstitute active nitrate reductase (NR) with apoenzyme from the Neurospora crassa mutant nit-1 was found mostly bound to a carrier protein (CP). This protein is scarce in the algal free extracts and has been purified 520-fold. MoCoCP is a protein of 64 kDa with subunits of 16.5 kDa and an isoelectric point of 4.5. In contrast to free MoCo, MoCo bound to CP was remarkably protected against inactivation under both aerobic conditions and basic pH. MocoCP transferred active MoCo to apoNR in vitro without addition of molybdate, though reconstituted activity was 20% higher in the presence of molybdate. Incubation with tungstate specifically inhibited MoCoCP activity but had no effect on the activity of free MoCo released from milk xanthine oxidase. MoCoCP did not charge molybdate unless in the presence of N. crassa extracts. Our data support that MoCoCP stabilizes MoCo in an active form charged with molybdate to provide MoCo to apomolybdoenzymes.

The regulation of flowering time by daylength in Arabidopsis.

We are studying several Arabidopsis mutants that show altered regulation of flowering time in response to daylength. One of the mutations we are studying, constans, delays flowering under long days but has no effect under short days. Analysis of the expression of the CONSTANS gene, and modification of its expression in transgenic plants, suggests that this gene promotes flowering in response to long days and that the delay in flowering that occurs in wild-type plants under short days is at least in part due to regulation of CONSTANS gene transcription. We describe genetic approaches that we are taking to identify genes that act in the same genetic pathway as CONSTANS, and in particular the relationship between CONSTANS and two other genes that we are studying. These are LATE ELONGATED HYPOCOTYL, for which we have a dominant mutant allele that causes late flowering, and EARLY SHORT DAYS 4, whose inactivation causes early flowering. In addition to their effects on flowering time, the over-expression of CONSTANS and the inactivation of EARLY SHORT DAYS 4 cause the Arabidopsis shoot to become determinate and therefore to terminate development prematurely. This phenotype is discussed in light of other genes that have previously been shown to be required to maintain indeterminate development of the shoot.

Activation of floral meristem identity genes in Arabidopsis.

The Arabidopsis floral meristem-identity genes APETALA1 (AP1) and LEAFY (LFY) confer floral identity on developing floral primordia, whereas TERMINAL FLOWER (TFL) is required to repress their expression within shoot and inflorescence meristems. LFY and AP1 are expressed in floral primordia in response to environmental conditions, such as day length, which regulate the onset of flowering, and presumably also in response to the action of genes that influence flowering time. However, the relationship between these flowering-time genes and the floral meristem-identity genes has been difficult to assess because flowering time is determined by several interacting genetic pathways. Here we describe a method to regulate expression of the flowering-time gene CONSTANS (CO) and demonstrate that CO expression is sufficient to trigger flowering, irrespective of day length. In response to CO expression, transcription of LFY and TFL is initiated rapidly, whereas transcription of AP1 occurs much later. We propose that CO acts within a genetic pathway that is sufficient to activate LFY and TFL transcription, but that rapid activation of AP1 requires an additional pathway.

Halotolerance of the Phototrophic Bacterium Rhodobacter capsulatus E1F1 Is Dependent on the Nitrogen Source.

Phototrophic growth of the moderate halotolerant Rhodobacter capsulatus strain E1F1 in media containing up to 0.3 M NaCl was dependent on the nitrogen source used. In these media, increased growth rates and growth levels were observed in the presence of reduced nitrogen sources such as ammonium and amino acids. When the medium contained an oxidized nitrogen source (dinitrogen or nitrate), increases in salinity severely inhibited phototrophic growth. However, the addition of glycine betaine promoted halotolerance and allowed the cells to grow in 0.2 M NaCl. Inhibition of diazotrophic growth by salinity was due to a decrease in nitrogenase activity which was no longer synthesized and reversibly inactivated, both effects being alleviated by the addition of glycine betaine. In R. capsulatus E1F1, inhibition of cell growth in nitrate by salt was due to a rapid inhibition of nitrate uptake, which led to a long-term decrease in nitrate reductase activity, probably caused by repression of the enzyme. Addition of glycine betaine immediately restored nitrate uptake, but the recovery of nitrate reductase activity required several hours. Neither ammonium uptake nor ammonium assimilation through the glutamine synthetase-glutamate synthase pathway was affected by NaCl.