Differential Gene Expression During Floral Transition in Pineapple
Pineapple (Ananas comosus var. comosus) and ornamental bromeliads are commercially induced to flower by treatment with ethylene or its analogs. The apex is transformed from a vegetative to a floral meristem and shows morphological changes in 8 to 10 days, with flowers developing 8 to 10 weeks later. During eight sampling stages ranging from 6 hours to 8 days after treatment, 7,961 genes were found to exhibit differential expression (DEG) after the application of ethylene. In the first 3 days after treatment, there was little change in ethylene synthesis or in the early stages of the ethylene response. Subsequently, three ethylene response transcription factors (ERTF) were up-regulated and the potential gene targets were predicted to be the positive flowering regulator CONSTANS (CO), a WUSCHEL gene, two APETALA1/FRUTFULL (AP1/FUL) genes, an epidermal patterning gene and a jasmonic acid synthesis gene. We confirm that pineapple has lost the flowering repressor FLOWERING LOCUS C. At the initial stages, the SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) was not significantly involved in this transition. Another WUSCHEL gene and a PHD homeobox transcription factor, though not apparent direct targets of ERTF, were up-regulated within a day of treatment, their predicted targets being the up-regulated CO, auxin response factors, SQUAMOSA, and histone H3 genes with suppression of abscisic acid response genes. The FLOWERING LOCUS T (FT), TERMINAL FLOWER (TFL), AGAMOUS-like APETELAR (AP2) and SEPETALA (SEP) increased rapidly within 2 to 3 days after ethylene treatment. Two FT genes were up-regulated in the apex and not the leaf bases after treatment, suggesting that transport did not occur. These results indicated that the ethylene response in pineapple and possibly most bromeliads acts directly to promote the vegetative to flower transition via APETALA1/FRUITFULL (AP1/FUL) and its interaction with SPL, FT, TFL, SEP and AP2. Overall design: A uniform plot with pineapple plants weighing 1.5 to 2 kg was selected in a commercial field (Cultivar 'MD1', PRI 73-50) at Dole Plantation, Wahiawa, Hawaii (21° 31' 52.6 N; -158° 03' 35.3 W) 6 weeks before commercial forcing. The plot was divided into three replications with two treatments (control and treated) and ~50 plants in each treatment block. Water (10 ml) as the control treatment or ethephon (50 mg ai in 10 mL) (Ethrel, Rhone-Poulenc, AG Company, North Carolina) as the flower induction treatment were injected into the center of the plant between 7:00 and 7:20 AM. At 8:30 to 8:45 am, three plants were harvested from each replication in the water control and ethephon-treated plants at each sampling time after treatment. Four grams of the "D" leaf base (most recently matured leaf) and the other leaves of the uprooted plants were trimmed in the field from the cut stem. The trimmed leaf bases and trimmed stem were chilled and immediately returned to the laboratory on ice at 10 AM for further processing. The bases of the trimmed leaves were removed from the apex of the stem to expose the stem apex, and <1 g of the apex was taken from each stem and frozen in liquid nitrogen. This initial sampling was completed by 2:00 PM and reported here as 6 hours after ethephon or water control treatment. Subsequent apex and 'D' leaf samples were taken 1, 2, 3, 4, 5, 6 and 8 days after treatment between 8:00 and 8:30 AM and processed by noon in the laboratory. Leaf bases were included as a control to allow comparison with non-apex tissue, since leaf primordia remained on the excised apex. All samples were stored at -80 ° C until ground into powder in liquid nitrogen for RNA extraction. Total RNA was extracted from the apex and leaf bases using the Qiagen RNeasy Plant Mini Kit (Qiagen, #74904) following the manufacturer's protocol. DNA was removed with the DNA-free DNA Removal Kit (Life Technologies, #AM1906M). Three biological replicates were sequenced for each sampling stage with three apices or leaf bases in each replication. Total RNA (2 µg) was used for the preparation of the mRNA-Seq library using the TruSeq Stranded mRNA LT kit (Illumina, USA) according to the manufacturer's protocol. The size of the RNA-Seq library was evaluated by electrophoresis (1 µl of sample + 1 µl of loading dye 6X, 1% Agarose, TBE 1X Buffer, 30 minutes, 60V) and 1 µl of sample was used for quantification with a Qubit Fluorometer (Invitrogen, USA) using the DNA HS assay kit. The multiplexed pooled libraries were sequenced on Illumina HiSeq4000 with a 50 nt read length. Small RNA libraries were prepared from total RNA using the NEBNext Multiplex Small RNA Sample Kit for Illumina (E7300, NEB, Ipswich, MA) following the manufacturer's protocol. The libraries were quantified by qPCR and sequenced in each lane with a read length of 50 nt.