Characterization of the response of the Arabidopsis response regulator gene family to cytokinin.

We examined the expression of a family of Arabidopsis response regulators (ARR) and found that the steady-state levels of RNA for most are elevated very rapidly by cytokinin. Using nuclear run-on assays we demonstrated that this increase in ARR transcript levels in response to cytokinin is due, at least in part, to increased transcription. The start site of transcription for the ARR5 gene was identified using primer extension analysis. A DNA fragment comprised of 1.6 kb upstream of the ARR5 transcript start site conferred cytokinin-inducible gene expression when fused to a beta-glucuronidase reporter, confirming that the transcription rate of ARR5 is elevated by cytokinin. This reporter construct was also used to examine the spatial pattern of ARR5 expression. The highest levels of expression were observed in the root and shoot apical meristems, at the junction of the pedicle and the silique, and in the central portion of mature roots. The expression of ARR5 in the apical meristems was confirmed by whole mount in situ analysis of seedlings and is consistent with a role for cytokinin in regulating cell division in vivo.

The RCN1-encoded A subunit of protein phosphatase 2A increases phosphatase activity in vivo.

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine-specific protein phosphatase, comprises a catalytic C subunit and two distinct regulatory subunits, A and B. The RCN1 gene encodes one of three A regulatory subunits in Arabidopsis thaliana. A T-DNA insertion mutation at this locus impairs root curling, seedling organ elongation and apical hypocotyl hook formation. We have used in vivo and in vitro assays to gauge the impact of the rcn1 mutation on PP2A activity in seedlings. PP2A activity is decreased in extracts from rcn1 mutant seedlings, and this decrease is not due to a reduction in catalytic subunit expression. Roots of mutant seedlings exhibit increased sensitivity to the phosphatase inhibitors okadaic acid and cantharidin in organ elongation assays. Shoots of dark-grown, but not light-grown seedlings also show increased inhibitor sensitivity. Furthermore, cantharidin treatment of wild-type seedlings mimics the rcn1 defect in root curling, root waving and hypocotyl hook formation assays. In roots of wild-type seedlings, RCN1 mRNA is expressed at high levels in root tips, and accumulates to lower levels in the pericycle and lateral root primordia. In shoots, RCN1 is expressed in the apical hook and the basal, rapidly elongating cells in etiolated hypocotyls, and in the shoot meristem and leaf primordia of light-grown seedlings. Our results show that the wild-type RCN1-encoded A subunit functions as a positive regulator of the PP2A holoenzyme, increasing activity towards substrates involved in organ elongation and differential cell elongation responses such as root curling.

A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis.

The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

Structure of a functional geranylgeranyl pyrophosphate synthase gene from Capsicum annuum.

A geranylgeranyl pyrophosphate synthase (GGPPS) gene from Capsicum annuum (bell pepper) was cloned. The nucleotide sequence shows that this gene, like the capsanthin/capsorubin gene but unlike the phytoene synthase gene from C. annuum, is not interrupted by an intron. Southern blot analysis of C. annuum genomic DNA suggests the presence of a single gene highly similar to the cDNA and also of additional related sequences. The present data suggest that this cloned gene is functional.

Structure and expression of two plant genes encoding chromoplast-specific proteins: occurrence of partially spliced transcripts.

The genes encoding fibrillin and capsanthin-capsorubin synthase are specifically expressed during fruit ripening in Capsicum annuum, leading to the accumulation of these two proteins in chromoplasts. Here, we report for the first time the cloning of genomic DNA fragments encoding these two enzymes, as well as DNA fragments containing upstream regions which are potentially involved in the regulation of the expression of these genes. While the capsanthin-capsorubin synthase gene is uninterrupted, the fibrillin gene is interrupted by two introns, the first one being inefficiently spliced. Occurrence of unspliced transcripts is apparently not related to a post-transcriptional mechanism controlling the synthesis of fibrillin or an alternative polypeptide. This work provides tools for studies on gene activation and intron splicing in plants.

Fibril assembly and carotenoid overaccumulation in chromoplasts: a model for supramolecular lipoprotein structures.

Chromoplast development in ripening bell pepper fruits is characterized by a massive synthesis of carotenoid pigments, resulting in their distinctive red color. We have shown that 95% of these pigments accumulate in chromoplasts in specific lipoprotein fibrils. In addition to carotenoids, purified fibrils contain galactolipids, phospholipids, and a single, 32-kD protein, designated fibrillin, which has antigenically related counterparts in other species. Fibrils were reconstituted in vitro when purified fibrillin was combined with carotenoids and polar lipids in the same stoichiometric ratio found in fibrils in vivo. Antibodies directed against fibrillin were used to isolate a fibrillin cDNA clone and, in immunological studies, to follow its accumulation during the chloroplast-to-chromoplast transition under different conditions. A model for fibril architecture is proposed wherein carotenoids accumulate in the center of the fibrils and are surrounded by a layer of polar lipids, which in turn are surrounded by an outer layer of fibrillin. Topological analysis of purified fibrils verified this structure. Collectively, these results suggest that the process of fibril self-assembly in chromoplasts is an example of a general phenomenon shared among cells that target excess membrane lipids into deposit structures to avoid their destabilizing or toxic effects. In addition, we have shown that abscisic acid stimulates this phenomenon in chromoplasts, whereas gibberellic acid and auxin delay it.