Giorgio Morelli

A novel class of plant proteins containing a homeodomain with a closely linked leucine zipper motif.

The homeobox, a 183 bp DNA sequence element, was originally identified as a region of sequence similarity between many Drosophila homeotic genes. The homeobox codes for a DNA‐binding motif known as the homeodomain. Homeobox genes have been found in many animal species, including sea urchins, nematodes, frogs, mice and humans. To isolate homeobox‐containing sequences from the plant Arabidopsis thaliana, a cDNA library was screened with a highly degenerate oligonucleotide corresponding to a conserved eight amino acid sequence from the helix‐3 region of the homeodomain. Using this strategy two cDNA clones sharing homeobox‐related sequences were identified. Interestingly, both of the cDNAs also contain a second element that potentially codes for a leucine zipper motif which is located immediately 3′ to the homeobox. The close proximity of these two domains suggests that the homeodomain‐leucine zipper motif could, via dimerization of the leucine zippers, recognize dyad‐symmetrical DNA sequences.

The Athb-1 and -2 HD-Zip domains homodimerize forming complexes of different DNA binding specificities.

The Arabidopsis Athb‐1 and −2 proteins are characterized by the presence of a homeodomain (HD) with a closely linked leucine zipper motif (Zip). We have suggested that the HD‐Zip motif could, via dimerization of the leucine zippers, recognize dyad‐symmetric DNA sequences. Here we report an analysis of the DNA binding properties of the Athb‐1 homeodomain‐leucine zipper (HD‐Zip‐1) domain in vitro. DNA binding analysis performed using random‐sequence DNA templates showed that the HD‐Zip‐1 domain, but not the Athb‐1 HD alone, binds to DNA. The HD‐Zip‐1 domain recognizes a 9 bp dyad‐symmetric sequence [CAAT(A/T)ATTG], as determined by selecting high‐affinity binding sites from random‐sequence DNA. Gel retardation assays demonstrated that the HD‐Zip‐1 domain binds to DNA as a dimer. Moreover, the analysis of the DNA binding activity of Athb‐1 derivatives indicated that a correct spatial relationship between the HD and the Zip is essential for DNA binding. Finally, we determined that the Athb‐2 HD‐Zip domain recognizes a distinct 9 bp dyad‐symmetric sequence [CAAT(G/C)ATTG]. A model of DNA binding by the HD‐Zip proteins is proposed.

The Arabidopsis Athb-8, -9 and -14 genes are members of a small gene family coding for highly related HD-ZIP proteins.

We report the isolation and characterization of two Arabidopsis homeobox genes highly related to the Athb-8 gene. The full-length cDNAs encode proteins of 841 and 852 amino acids which we have designated Athb-9 and -14, respectively. Athb-8, -9 and -14 are members of a small family of HD-Zip proteins (HD-ZIP III) characterized by a HD-Zip motif confined to the N-terminus of the polypeptide. The spatial organization of the HD-Zip domain of Athb-8, -9 and -14 is different from that of the Athb-1 (a member of the HD-ZIP I family) and Athb-2 (a member of the HD-ZIP II family) HD-Zip domains. DNA binding analysis performed with random-sequence DNA templates showed that the Athb-9 HD-Zip (HD-Zip-9) domain, but not the Athb-9 HD alone, binds to DNA. The HD-Zip-9 domain recognizes a 11 bp pseudopalindromic sequence (GTAAT(G/C)ATTAC), as determined by selecting high-affinity binding sites from random-sequence DNA. Moreover, gel retardation assays demonstrated that the HD-Zip-9 domain binds to DNA as a dimer. These data support the notion that the HD-ZIP III domain interacts with DNA recognition elements in a fashion similar to the HD-ZIP I and II domains.

Expression dynamics of the pea rbcS multigene family and organ distribution of the transcripts

We have determined the nucleotide sequence of two members (rbcS‐3A and ‐3C) of the pea nuclear gene family encoding the small subunit (rbcS) of ribulose‐1,5‐bisphosphate carboxylase. Both rbcS‐3A and ‐3C are interrupted by two introns located at the same positions as those of the other three pea rbcS genes. Compared with the other pea rbcS genes the rbcS‐3C gene has the most divergent 5′‐ and 3′‐flanking sequences while the rbcS‐3A gene has a larger and highly divergent intron 1. All five pea rbcS genes are conserved in their coding regions but show considerable sequence differences in their 3′‐untranslated portion. The 3′ sequence divergence of the rbcS genes has allowed us to use S1 nuclease mapping procedures to compare their expression levels in different organs and during light induction. All the rbcS genes are differentially expressed in various organs of the pea plants; moreover, specific rbcS transcripts are under‐represented in seeds and petals. In leaves there is a 10‐fold difference between the highest and lowest specific rbcS transcript levels. By quantitating the distribution of rbcS transcripts during light, phytochrome and blue light induction of immature (etiolated), and mature (green), pea leaves, we show that the genes are differentially activated during leaf development.

Light and shade in the photocontrol of Arabidopsis growth

Plants have evolved sophisticated sensing mechanisms that operate through phytochromes, perceiving changes in the red:far-red ratio, which trigger morphological changes to avoid shade. The shade-avoidance response essentially redirects resources and growth potential from the leaf and storage organs into increased extension growth to optimize light capture by plants. Recent studies implicate ATHB-2, a homeodomain-leucine zipper transcription factor, as a regulator of shade-avoidance responses and establish a strong link between this factor and auxin signaling. The action of ATHB-2 is likely to generate changes in auxin distribution that produce distinct but coordinated effects on different cell types across the plant. Future studies should highlight how polarity of auxin transport is altered in response to light-quality changes.

Photoregulated expression of a pea rbcS gene in leaves of transgenic plants

A 2.4‐kb pea genomic fragment, containing a member (rbcS‐E9) of the multigene family encoding the small subunit (rbcS) of ribulose‐1,5‐bisphosphate carboxylase, was inserted into a non‐oncogenic, Ti‐plasmid vector and introduced into the genomes of Petunia hybrida (Mitchell) and Nicotiana tabacum (SR1) plants by in vitro transformation. Petunia and tobacco plants containing the introduced pea rbcS‐E9 gene were regenerated from protoplasts. In these transgenic plants the rbcS‐E9 gene is transcribed accurately using its own promoter and its expression is light‐induced and organ‐specific. A deletion mutant with 352 bp of 5′‐upstream sequence still retains photoinducibility and leaf‐specific expression. Clonal analysis of independent transgenic petunia plants revealed that chromosomal positions in the recipient plant genome affect the quantitative but not qualitative aspects of rbcS‐E9 expression.

Identification of Distinct Families of HD-ZIP Proteins in Arabidopsis Thaliana

Many of the regulatory genes involved in the control of development share a common sequence element of 180 bp, the homeobox (HB), which encodes a 60 amino acid motif, the homeodomain (HD; Scott et al., 1989; Gehring et al., 1990). The amino acid sequences of known HDs are conserved in evolution from yeast to higher vertebrates (Scott et al., 1989). Recently HB genes have been identified in two plant species, maize (Vollbrecht et al., 1991; Bellmann and Werr, 1992) and Arabidopsis (Ruberti et al., 1991). Despite the differences in plant and animal development the discovery of homeobox genes in plants suggests that fundamental regulatory mechanisms that control development may be shared among all higher eukaryotes. The analysis of the maize kn1 mutants has shown that ectopic expression of the knotted gene profoundly affects leaf development, suggesting that HD proteins in plants might be involved in differentiation and/or developmental control as they are in animals (Hake, 1992).

The Interplay of Light and the Circadian Clock (Independent Dual Regulation of Clock-Controlled Gene ccg-2(eas)

Ambient light is the major agent mediating entrainment of circadian rhythms and is also a major factor influencing development and morphogenesis. We show that in Neurospora crassa the expression of clock-controlled gene 2 (ccg-2), a gene under the control of the circadian clock and allelic to the developmental gene easy wettable (eas), is regulated by light in wild-type strains. Light elicits a direct and important physiological effect on ccg-2(eas) expression as demonstrated using several mutant Neurospora strains. In white collar mutants (wc-1 and wc-2) that are “blind” to blue light, ccg-2(eas) mRNA shows no variation following illumination with saturating light. By contrast, ccg-2(eas) mRNA is photoinduced in clock-null strains such as frequency9 (bd;frq9). The results in the clock mutants show that an intact circadian oscillator is not required for light induction of ccg-2(eas). Thus, ccg-2(eas) is subject to a dual regulation that involves separable regulation by light and circadian rhythm.

Arabidopsis HD-Zip II transcription factors control apical embryo development and meristem function

The Arabidopsis genome encodes ten Homeodomain-Leucine zipper (HD-Zip) II proteins. ARABIDOPSIS THALIANA HOMEOBOX 2 (ATHB2), HOMEOBOX ARABIDOPSIS THALIANA 1 (HAT1), HAT2, HAT3 and ATHB4 are regulated by changes in the red/far red light ratio that induce shade avoidance in most of the angiosperms. Here, we show that progressive loss of HAT3, ATHB4 and ATHB2 activity causes developmental defects from embryogenesis onwards in white light. Cotyledon development and number are altered in hat3 athb4 embryos, and these defects correlate with changes in auxin distribution and response. athb2 gain-of-function mutation and ATHB2 expression driven by its promoter in hat3 athb4 result in significant attenuation of phenotypes, thus demonstrating that ATHB2 is functionally redundant to HAT3 and ATHB4. In analogy to loss-of-function mutations in HD-Zip III genes, loss of HAT3 and ATHB4 results in organ polarity defects, whereas triple hat3 athb4 athb2 mutants develop one or two radialized cotyledons and lack an active shoot apical meristem (SAM). Consistent with overlapping expression pattern of HD-Zip II and HD-Zip III gene family members, bilateral symmetry and SAM defects are enhanced when hat3 athb4 is combined with mutations in PHABULOSA (PHB), PHAVOLUTA (PHV) or REVOLUTA (REV). Finally, we show that ATHB2 is part of a complex regulatory circuit directly involving both HD-Zip II and HD-Zip III proteins. Taken together, our study provides evidence that a genetic system consisting of HD-Zip II and HD-Zip III genes cooperates in establishing bilateral symmetry and patterning along the adaxial-abaxial axis in the embryo as well as in controlling SAM activity.

Functional identification of al-3 from Neurospora crassa as the gene for geranylgeranyl pyrophosphate synthase by complementation with crt genes, in vitro characterization of the gene product and mutant analysis.

In this work the Neurospora crassa al-3 gene function was determined. Geranylgeranyl pyrophosphate (GGPP) synthase activity was measured in al-2 FGSC 313 and al-3 RP100 FGSC 2082 mutant strains by in vitro synthesis methods. This experiment showed that al-3 RP100 mutant expresses a reduced GGPP synthase activity. The mutated al-3 gene was cloned and sequenced; a single missense mutation was found changing serine into asparagine. Genetic complementation was performed by Escherichia coli transformation, with clusters of crt genes from Erwinia uredovora. Carotenoid accumulation was observed in E. coli transformants when the N. crassa al-3 gene substitutes the GGPP synthase gene (crtE) in the carotenogenic crt cluster. Cell-free studies with E. coli transformants gave direct evidence of the function of the al-3 protein as GGPP synthase and indicated that a short-chain prenylpyrophosphate, such as dimethylallyl pyrophosphate, is the genuine substrate.