The tomato (Lycopersicon esculentum, Mill.) mutant diageotropica (dgt) exhibits biochemical, physiological, and morphological abnormalities that suggest the mutation may have affected a primary site of auxin perception or action. We have compared two aspects of the auxin physiology of dgt and wild-type (VFN8) seedlings: auxin transport and cellular growth parameters. The rates of basipetal indole-3-acetic acid (IAA) polar transport are identical in hypocotyl sections of the two genotypes, but dgt sections have a slightly greater capacity for IAA transport. 2,3,5-Triiodobenzoic acid and ethylene reduce transport in both mutant and wild-type sections. The kinetics of auxin uptake into VFN8 and dgt sections are nearly identical. These results make it unlikely that an altered IAA efflux carrier or IAA uptake symport are responsible for the pleiotropic effects resulting from the dgt mutation. The lack of auxin-induced cell elongation in dgt plants is not due to insufficient turgor, as the osmotic potential of dgt cell sap is less (more negative) than that of VFN8. An auxininduced increase in wall extensibility, as measured by the Instron technique, only occurs in the VFN8 plants. These data suggest dgt hypocotyls suffer a defect in the sequence of events culminating in auxin-induced cell wall loosening.The analysis of mutants is a classical approach to determining the physiological relevance of certain proteins and elucidating biochemical pathways. This tactic can be used to understand better the mechanism of auxin action. A tomato (Lycopersicon esculentum, Mill.)

ABSTRACT

Photooxidative destruction of chloroplasts by exposure of norflurazon-treated cucumber (Cucumis sativus L.) seedlings to white light leads to reduced levels of the nuclear-encoded, peroxisomal enzyme hydroxypyruvate reductase. The partial reduction in hydroxypyruvate reductase activity under photooxidative conditions is accompanied by reductions in levels of hydroxypyruvate reductase protein and transcript. The low level of hydroxypyruvate reductase gene expression in the dark is not affected by norflurazon, and nonphotooxidizing far-red light is able to induce significant increases in hydroxypyruvate reductase expression even in the presence of norflurazon. We conclude that intact plastids are required for maximal expression of hydroxypyruvate reductase in the light and that the plastids affect hydroxypyruvate reductase gene expression at a pretranslational level.

Transcription of the cucumber hpr-A gene is responsive to cytokinin and light. To investigate the molecular basis for transcriptional regulation by cytokinin, we have identified DNA sequences and proteins that may be involved in the regulation of hpr-A gene expression. Transient expression assays in etiolated cucumber cotyledons indicate that the 315 bp fragment (-382 to -67) contains sequences necessary for cytokinin responsiveness of the luciferase reporter gene. Band shift assays detected cytokinin-enhanced and -reduced protein binding sites in a 97 bp fragment (-382 to -285) upstream of the hpr-A gene. DNase I footprinting identified two protein-protected sites, a 15 bp sequence, 5'-AAATGACGAAAATGC-3', that contains an as-1 TGACG motif found in other plant promoters, and a 13 bp sequence, 5'-AAGATTGATTGAG-3', of unknown function. Two-dimensional band shift analysis of the cytokinin-responsive DNA protein complex revealed the presence of six DNA protein interactions. Band shift assays showed that cytokinin and light have different effects on the interaction of nuclear proteins to the 97 bp fragment of the hpr-A gene. These data suggest that cytokinin and light do not share identical signal transduction pathways in regulating hpr-A gene expression.

The 5'- and 3'-flanking regions of HPRA, a cucumber gene that encodes hydroxypyruvate reductase, were evaluated for regulatory activity with respect to light responsiveness and organ specificity. To define the functional regions of the 5'-flanking region of HPRA, a series of deletions was generated and the remaining portions fused to the beta-glucuronidase (GUS) reporter gene (uidA) containing a minimal 35S promoter truncated at -90. The region from -66 to +39 was found to be necessary for light-regulated expression of the uidA reporter gene, while the region from -382 to -67 was found to be necessary for its leaf-specific expression. Further deletion of the HPRA 5' flanking region to -590 resulted in high levels of root expression, suggesting the presence of a negative regulatory element responsible for silencing root expression of the HPRA gene between -590 and -383. The 3'-flanking region of the HPRA gene downstream of the polyadenylation site contains several sequence motifs resembling regulatory elements present in the promoters of several light-responsive genes. An 823 bp portion of the HPRA 3'-flanking region containing these putative regulatory elements enhanced GUS expression in leaves when placed downstream of the uidA reporter gene in the forward orientation, but not in the reverse orientation. When placed 5' of the -90 35S promoter, the 823 bp fragment enhanced slightly, independently of orientation, the root tip-specific expression pattern intrinsic to the -90 35S promoter, indicating that in some cases this region can act as a transcriptional enhancer.