The Functions Of A Phytochrome-regulated B-BOX Type Zinc Finger Protein Gene In Rice Growth And Development

In the previous work, we compared gene profiles between wild-type (WT) andphyB-deficiency mutants using the GeneChip. One candidate gene encoding B-boxtype zinc finger protein was identified to be down-regulated by phytochrome B(phyB)-mediated red light signal. Phylogenetic tree analysis revealed that this proteinwas classified into the same subgroup as Arabidopsis BBX22protein. Thus we namedthe gene as OsBBX22b.In order to discover the roles of OsBBX22b gene in rice growth, in this study, weanalyzed the expression patterns of OsBBX22b, subcellular localization of OsBBX22b,as well as identified the proteins interacting with OsBBX22b. We also producedOsBBX22b-overexpression (OsBBX22b-OX) lines and characterized the growth anddevelopment of transgenic rice plants. The results we obtained are as followed:(1) The level of OsBBX22b transcripts was very high in leaves, especially in theflag leaf. Moreover, OsBBX22b transcripts were regulated by photoperiod and circadianclock. These results suggest that OsBBX22b is likely to be involved in photoperiodicresponses in rice. We analyzed the effects of exogenous hormones JA, ABA, and abioticstress (including PEG and NaCl) on OsBBX22b transcripts. The results suggest that JAmay inhibit OsBBX22b gene expression. Exogenous ABA transiently inducedOsBBX22b gene expression, whereas drought and salt stress transiently inhibitedOsBBX22b expression.(2) The OsBBX22b protein located in the nucleus by analyzing the the subcellularlocalization in rice protoplast. A modified yeast one-hybrid analyses indicated thatOsBBX22b had a transcriptional activation potential, at least in yeast.(3) The preliminary results from the yeast two-hybrid experiments revealed thatOsBBX22b had interactions with seven Arabidopsis transcription factors.(4) OsBBX22b-OX seedlings were less sensitive to red and far-red light pulse thanWT with regards to photoinhibition of coleoptile elongation, indicating that OsBBX22bprobably acts as a negative regulator of photomorphogenesis in rice seedlings.(5) OsBBX22b-OX seedlings were sensitive to exogenous ABA. Moreover,overexpression of OsBBX22b did not affect the expression of genes related to ABAmetabolism by RT-PCR analysis. These results suggest that OsBBX22b probably affects the ABA response pathway.(6) The plant heights of OsBBX22b-OX were significantly shorter than the WTthroughout the different developmental stages. The lengths of the first, second, fourthinternodes of the transgenic rice were shorter than that of WT, while the third and fifthinternodes length had no significant difference. These results suggest thatoverexpression of OsBBX22b suppresses the elongation of the first, second, fourthinternode.(7) OsBBX22b-OX lines flowered later than the WT in the natural long-dayconditions and greenhouse short-day conditions, suggesting that overexpression ofOsBBX22b delayed rice flowering time. QRT-PCR analyses indicated that the rhythmicprofiles of two circadian-regulated genes, OsLHY and OsPRR1, in OsBBX22b-OX linesand the WT were indistinguishable from the WT, suggesting that OsBBX22b gene actsin output pathways downstream of the circadian clock. We further analyzed theexpression levels of two florigen genes (Hd3a and RFT1) as well as their upstreamgenes (Hd1and Ehd1) in60-day plants of OsBBX22b-OX and the WT. Our resultsindicate that OsBBX22b suppressed the Hd3a and RFT1expression, but did not affectthe upstream gene Hd1and Ehd1. Therefore, OsBBX22b regulated rice photoperiodicflowering via another way different from Hd1and RFT1.(8) OsBBX22b-OX lines exhibited reduced leaf length, panicle length, leaf area,grains per plant, the number of florets, panicle weight, but the increased1000-grainweight, compared with the WT plants. However, the leaf angle and chlorophyllaccumulation at seedling stage have no difference in OsBBX22b-OX lines from the WT.(9) In addition, comparing the gene profiles between OsBBX22b-OX and WTrevealed that OsBBX22b up-regulated the expression of185genes, among which22genes encode putative transcription factors, and down-regulated116genes. Amongthem, at least six genes related to JA pathway, OsbHLH148, OsJAZ1, OsJAZ3, OsJAZ7,OsJAZ11, and OsJAZ13, were up-regulated in OsBBX22b–OX lines. These resultssuggest that OsBBX22b probably play important roles in JA pathway in rice.In conclusion, our findings demonstate the important roles of OsBBX22b gene inrice growth and development.