Prateek Tripathi, Marcela Carvallo, Elizabeth E. Hamilton, Sasha Preuss, and Steve A. Kay

Significance

Clock genes have been shown to be important in regulating many key agronomic traits. Therefore, identifying new players in this interconnected clock network will provide novel strategies toward developing new crop varieties. Our study identifies CONSTANS-LIKE 3 (COL3) as a critical protein-binding partner for B-BOX32 (BBX32) action in Arabidopsis. The discovery of the interaction with COL3 provides molecular clues as to how BBX32 exerts its effects on growth and yield. It also implicates COL3 as an integral protein-binding partner that can be used in combination with BBX32 for increased productivity. This regulatory pathway could be applied as an efficient strategy for genetic manipulation in crops for increased agricultural productivity.

Abstract

Plants have the ability to respond to seasonal environmental variations by monitoring day length to initiate flowering. The transition from vegetative to the reproductive stage is the critical developmental switch in flowering plants to ensure optimal fitness and/or yield. It has been previously reported that B-BOX32 (BBX32) has the potential to increase grain yield when ectopically expressed in soybean. In the present study, we performed a detailed molecular characterization of the Arabidopsis B-box domain gene BBX32. We showed that the circadian clock in Arabidopsis regulates BBX32 and expressed in the early morning. To understand the molecular mechanism of BBX32 regulation, we performed a large-scale yeast two-hybrid screen and identified CONSTANS-LIKE 3 (COL3)/BBX4 as one of its interacting protein partners. Using different genetic and biochemical assays, we have validated this interaction and shown that COL3 targets FT in the presence of BBX32 to regulate the flowering pathway. Based on these findings, we hypothesized that this BBX32-COL3 module could be an additional regulatory mechanism affecting the reproductive development in Arabidopsis that could be translated to crops for increased agricultural productivity.

See: http://www.pnas.org/content/114/1/172.abstract.html?etoc

PNAS January 3 2017; vol.114; no.1: 172–177

Fig. 1.

Gene expression and phenotype analysis of BBX32. Relative gene expression analysis of BBX32 under diurnal conditions (12L:12D) (A) and free running conditions (LL) (B) at 22 °C. Wild-type (Col-0) seedlings were grown in 12L:12D for 7 d (A) and 12L:12D for 6 d and transferred to continuous light (LL) and harvested 24–68 h later (B). Error bars represent the SEM of biological triplicates. Experiments were independently repeated three times, each time with two biological replicates per genotype. (C) BBX32-OX:LHY-LUC⁺ activity under LL. Six-day-old seedlings entrained in 12L:12D were monitored for 5–7 d under LL. Values are shown as mean ± SEM; n = 20. (D) Relative amplitude error of WT and BBX32-OX seedlings imaged for 5 d under LL conditions calculated by using FFT-NLLS; n = 20. (E) Relative mRNA expression of LHY in WT and BBX32-OX. Six-day-old WT and BBX32-OX seedlings were grown in 12L:12D and transferred to continuous light (LL) and harvested 24–68 h later. (F) BBX32-AMI:LHY-LUC⁺ activity under LL. Six-day-old seedlings entrained in 12L:12D were monitored for 5–7 d under LL. Values are shown as mean ± SEM; n = 20. Experiments were repeated three times independently.