Pablo A. Scolnik

Regulation of carotenoid biosynthesis during tomato development.

Phytoene synthase (Psy) and phytoene desaturase (Pds) are the first dedicated enzymes of the plant carotenoid biosynthesis pathway. We report here the organ-specific and temporal expression of PDS and PSY in tomato plants. Light increases the carotenoid content of seedlings but has little effect on PDS and PSY expression. Expression of both genes is induced in seedlings of the phytoene-accumulating mutant ghost and in wild-type seedlings treated with the Pds inhibitor norflurazon. Roots, which contain the lowest levels of carotenoids in the plant, have also the lowest levels of PDS and PSY expression. In flowers, expression of both genes and carotenoid content are higher in petals and anthers than in sepals and carpels. During flower development, expression of both PDS and PSY increases more than 10-fold immediately before anthesis. During fruit development, PSY expression increases more than 20-fold, but PDS expression increases less than threefold. We concluded that PSY and PDS are differentially regulated by stress and developmental mechanisms that control carotenoid biosynthesis in leaves, flowers, and fruits. We also report that PDS maps to chromosome 3, and thus it does not correspond to the GHOST locus, which maps to chromosome 11.

The water- and salt-stress-regulated Asr1 (abscisic acid stress ripening) gene encodes a zinc-dependent DNA-binding protein

Tomato (Lycopersicon esculantum) ASR1 (abscisic acid stress ripening protein), a small plant-specific protein whose cellular mode of action defies deduction based on its sequence or homology analyses, is one of numerous plant gene products with unknown biological roles that become over-expressed under water- and salt-stress conditions. Steady-state cellular levels of tomato ASR1 mRNA and protein are transiently increased following exposure of plants to poly(ethylene glycol), NaCl or abscisic acid. Western blot and indirect immunofluorescence analysis with anti-ASR1 antibodies demonstrated that ASR1 is present both in the cytoplasmic and nuclear subcellular compartments; approx. one-third of the total ASR1 protein could be detected in the nucleus. Nuclear ASR1 is a chromatin-bound protein, and can be extracted with 1 M NaCl, but not with 0.5% Triton X-100. ASR1, overexpressed in Escherichia coli and purified to homogeneity, possesses zinc-dependent DNA-binding activity. Competitive-binding experiments and SELEX (systematic evolution of ligands by exponential enrichment) analysis suggest that ASR1 binds at a preferred DNA sequence.

Tomato Asr1 mRNA and protein are transiently expressed following salt stress, osmotic stress and treatment with abscisic acid

Abstract The Asr1 cDNA clone was isolated by differential screening of tomato ( Lycopersicon esculentum Mill., cv. Ailsa Craig) ripening fruit cDNA library with cDNA prepared from RNA isolated from leaves of water-stressed vs. irrigated tomato seedlings. In this study the steady state levels of tomato Asr1 mRNA and protein were further investigated. Low levels of mRNA and protein were detected in the roots and shoots of hydroponically grown tomato plants. Application of NaCl or PEG to the growth medium resulted in an elevation of the steady state levels of both Asr1 mRNA and protein. This increase was transient, reaching a maximum 12–24 h after the application of the stress. The extent of the increase correlated with the severity of the stress. The similarity between the response of mRNA and protein to the stress suggests that the Asr1 gene is regulated mainly by RNA transcription or RNA stability. The response of Asr1 to water and salt stresses could be mimicked by treatment of the seedlings with ABA, suggesting that this hormone is involved in the mechanism of activation of the Asr1 gene by these abiotic stresses.

Genomic Nucleotide Sequence of Tomato Asr2, a Second Member of the Stress/Ripening-Induced Asr1 Gene Family

Gene expression in plants is regulated by developmental stage (for example, see Gillapsy et al., 1993; Meekswager, 1993; Thomas, 1993) and by response to the environment (Bray, 1991, 1993; Gurley et al., 1993; Quatrano et al., 1993; Winicov, 1993). Water stress is a major abiotic stress that triggers a massive change in gene expression (for recent reviews, see Bray, 1991, 1993). Iusem et al. (1993) recently isolated the tomato Asrl cDNA clone by differential screening of a tomato fruit cDNA library with cDNA prepared from irrigated versus water-stressed leaves. Asrl encodes a 13-kD basic protein, which is localized in the nucleus (Iusem et al., 1993). Genomic Southem analysis suggested that Asrl is a member of a small gene family (not shown). The Asrl cDNA sequence was used as a probe in screening of a tomato genomic library, and severa1 independent clones were isolated. We describe here genomic clone 18, which contains a second member of the Asr gene family, which we have designated Asr2 (Table I). The gene contains a 342-bp-long open reading frame interrupted by a 112-bp intron. As expected, the AT content of the intron is significantly higher than that of the coding sequence (77 versus 52%, respectively). Nucleotide sequences of the coding regions of Asrl and Asr2 show.82% identity. Thus, the Asr2 gene encodes a protein that is very homologous to that encoded by Asrl (78% identity and 83% similarity). Asr2 encodes a slightly basic 114-amino acid polypeptide (calculated isoelectric point = 7.34), and 55% of the residues are of charged amino acids. The most abundant residues are His (24 residues), Ala (14 residues), Lys (18 residues), and Glu (17 residues). The homology between ASRl and ASR2 proteins is restricted to the first 100 residues of the polypeptides (84% identity and 88% similarity). No homology was detected for the 15 and 12 residues in the carboxy terminus of the ASRl and ASR2 proteins, respectively. We have also studied the expression of the Asrl gene at both the mRNA and protein levels (H. Amitai-Zeigerson, unpublished data).

Regulation of carotenoid biosynthesis genes during plant development

Expression of the carotenoid biosynthesis genes PDS and PSY during development of tomato plants was studied by reverse transcriptase-polymerase chain reaction (RT-PCR). Results indicate that both stress and developmental signals control the expression of these genes. The PDS was mapped to the chromosome 3 of tomato.

Functional analysis and purification of enzymes for carotenoid biosynthesis expressed in photosynthetic bacteria.

Publisher Summary This chapter discusses methods for the inducible expression of carotenoid biosynthesis genes in Rhodobacter capsulatus and Escherichia coli along with novel procedures for the purification of phytoene desaturases. Carotenoid desaturases are membrane-bound enzymes in both bacteria and plants. The functional expression of Neurospora crassa and Glycine max (soybean) cDNAs coding for carotenoid desaturases in mutants of the photosynthetic bacterium Rhodobacter capsulatus have been achieved. Thus, it is logical to expect functional expression in R. capsulatus of carotenoid desaturase cDNAs from other species. In addition, it is possible that cDNAs coding for carotenoid enzymes other than desaturases could be expressed in R. capsulatus carotenoid mutants. Phytoene desaturases from various organisms catalyze a different number of desaturations. Rhodobacter capsulatus phytoene desaturase converts phytoene to neurosporene, a three-step desaturation reaction. Neurospora crassa Al-1 catalyzes six desaturations. Soybean Pds1 converts phytoene to ζ-carotene, a two-step desaturation reaction. Expression of al-1 in a phytoene-accumulating strain of R. capsulatus leads to the accumulations of lycopene and 3,4 dehydrolycopene. Expression of pds in this bacterial strain results in the accumulation of ζ-carotene. Lycopene, 3,4-dehydrolycopene, and ζ-carotene are not normally accumulated in R. capsulatus .