Functional Characterization of C3HC4-type RING Zinc Finger Protein Gene from Hot Pepper

Abstract

RING finger is a cysteine-rich domain (40–60 residues) in which cysteine and histidine residues ligated with two zinc ions in a cross-brace manner to stabilize the domain structure. RING finger proteins play a crucial role in diverse biological processes such as transcriptional activation, recombination of DNA, translational processes, signal transduction, programmed cell death, membrane association, and protein folding and assembly. RING finger proteins have also been implicated in various and fundamental functions in plant growth and development. Although, RING finger proteins have been closely and repeatedly involved in the development of multiple organisms, however, cases reported in plants are comparatively limited. Capsicum annuum RING Zinc Finger Protein 1 (CaRZFP1) is a C3HC4-type RING zinc finger protein gene previously isolated from a cDNA library of heat-stressed hot pepper. Expression of CaRZFP1 was also induced by diverse abiotic stresses including cold, dehydration and high salinity in hot pepper. Transcript induction kinetics of CaRZFP1 was distinct in different plant parts of hot pepper. In our previous work elucidating in vivo function of CaRZFP1, we transferred CaRZFP1 into tobacco (Nicotiana tabacum)

transgenic tobacco exhibited enhanced growth and tolerance to abiotic stresses. As further analysis of CaRZFP1 ectopic expression in a heterologous host plant, this study, I mobilized and constitutively overexpressed CaRZFP1 into lettuce (Lactuca sativa). In contrast to tobacco, transgenic lettuce exhibited poorer growth and delayed flowering compared with vector-only controls

characteristics included weakened leaf growth, shorter plant height, and stunted root growth. Thus, ectopic expression of CaRZFP1 caused pleiotropic developmental changes in the CaRZFP1-transgenic lettuce plants. In addition, I found a significant correlation between CaRZFP1 expression and the degree of diminished growth of CaRZFP1-transgenic lettuce plants. Starting from T2, I categorized transgenic lettuce lines according to CaRZFP1 transcript expression levels (low, medium, and high). The correlation between CaRZFP1 transcript level and a negative phenotypic effect was repeatedly maintained through the next generations of CaRZFP1-transgenic lettuce plants. Overall, CaRZFP1 expression impeded the growth and development of CaRZFP1-transgenic lettuce plants in a dose-dependent fashion. The weaken growth of CaRZFP1-transgenic lettuce plants continued to the late stage of development. At full growth, CaRZFP1-transgenic lettuce was shorter than vector-only plants. CaRZFP1-transgenic lettuce delayed in flowering and inflorescence size was smaller compared to vector-only plants. Flower size did not differ significantly between the transgenic and control lines, but the former head significantly fewer flowers per inflorescence. To examine the retarded growth of CaRZFP1-transgenic lettuce and the robust growth of CaRZFP1-transgenic tobacco at the cellular level, I analyzed leaf, stem, and root sections of both plants and compared them with vector-only controls. The cross sections of leaves and stems of CaRZFP1-transgenic lettuce and vector-only lettuce were not distinguishable in terms of morphology, cell size, or tissue organization. However, the development of endodermis and vascular bundles was significantly hampered by CaRZFP1 expression in transgenic lettuce roots. Remarkably, I observed a strong correlation, albeit negative, between the expression level of CaRZFP1 and the degree of disruption of internal root morphology. The overall root morphology and tissue patterns were preserved in CaRZFP1-transgenic tobacco roots. CaRZFP1 over-expression effect on the abiotic stress tolerance in CaRZFP1-transgenic lettuce plants was also examined, but no significant differences were observed between the CaRZFP1-transgenic lettuce and vector-only plants. To identify genes that might be involved in this phenotypic effect, transcriptome analyses on transgenic plants of both species were performed, uncovering dozens of genes that reflect the different outcomes between tobacco and lettuce. In particular, the strong negative correlation between CaRZFP1 expression and growth in lettuce helped us isolate genes most likely involved in the phenotypic differences. These included protein kinase, transcriptional factor, transporter protein, hormone and metabolism-related genes, and some unannotated genes. I separated the up- and down-regulated genes into two groups: Group 1 included genes with correlative changes in expression level among the four CaRZFP1-transgenic lettuce lines (#6, #14, #16 and #12), while Group 2 included only genes with significant expression-level changes mainly in line #12 (highest CaRZFP1 expression). To validate the transcriptome profiling results, nine genes were randomly selected for oligo-RNA blot analysis either significantly up- or down-regulated in transgenic lettuce lines. Oligo-RNA blot results showed that the trends of the differentially expressed genes were generally consistent across the two different approaches. Therefore, collectively these results showed that a gene with a specific function in one organism can yield completely different effects depending on the host species it is moved into and address concerns of unexpectedly different outcome of a gene in different genetic environments.