Differential Gene Expression During Floral Transition in Pineapple. 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.

菠萝（Ananas comosus var. comosus）与观赏凤梨在商业生产中可通过乙烯或其类似物处理诱导开花。植株茎尖会从营养生长状态向花分生组织转变，并在8~10天内出现形态变化，花朵则于8~10周后发育形成。在处理后6小时至8天的8个采样阶段中，共发现7961个基因在乙烯处理后呈现差异表达（differential expression, DEG）。处理后的前3天内，乙烯合成及乙烯响应早期阶段几乎无明显变化。随后，3个乙烯响应转录因子（ethylene response transcription factor, ERTF）被上调表达，其预测的潜在靶基因包括正向开花调控因子CONSTANS（CO）、1个WUSCHEL基因、2个APETALA1/FRUITFULL（AP1/FUL）基因、1个表皮模式形成基因以及1个茉莉酸合成基因。本研究证实菠萝已丢失开花抑制因子FLOWERING LOCUS C。在该转变的初始阶段，CONSTANS过表达抑制因子1（SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1, SOC1）未发挥显著作用。另有1个WUSCHEL基因与1个PHD同源框转录因子，虽并非ERTF的直接靶标，但在处理后1天内即被上调；其预测靶标包括上调的CO、生长素响应因子、SQUAMOSA以及组蛋白H3基因，并伴随脱落酸响应基因的抑制。FLOWERING LOCUS T（FT）、TERMINAL FLOWER（TFL）、AGAMOUS样APETALA2（AP2）与SEPALLATA（SEP）基因在乙烯处理后2~3天内快速上调表达。处理后，茎尖中存在2个FT基因的上调表达，而叶基中未检测到该现象，这表明未发生信号转运。上述结果表明，菠萝乃至多数凤梨属植物的乙烯响应可通过APETALA1/FRUITFULL（AP1/FUL）及其与SPL、FT、TFL、SEP及AP2的相互作用，直接促进营养生长向开花转变。实验设计：于商业催花前6周，在夏威夷州瓦伊阿瓦市多尔种植园（21°31′52.6″N；158°03′35.3″W）的商业大田内，选取株重1.5~2 kg的长势一致的菠萝植株，品种为‘MD1’（PRI 73-50）。该试验田划分为3个生物学重复，设置2种处理（对照组与处理组），每个处理区块约含50株植株。于上午7:00至7:20间，向植株中心注入10 mL无菌水作为对照处理，或注入10 mL含50 mg有效成分的乙烯利（ethephon，商品名Ethrel，罗门哈斯公司，北卡罗来纳州AG公司）作为开花诱导处理。于处理后各采样时间点的上午8:30至8:45间，从每个生物学重复的对照组与乙烯利处理组中各采收3株植株。将拔起的植株的‘D’叶基（即最新成熟的叶片）及其余叶片从切下的茎秆上剥离，每份取样量为4 g。剥离的叶基与茎秆置于冰上冷藏，并于上午10时即刻送回实验室进行后续处理。将剥离的叶基从茎尖处去除以暴露茎尖，从每株茎尖中取<1 g的组织，置于液氮中速冻保存。该首轮采样于下午2时前完成，对应乙烯利或水处理后6小时的采样点。后续的茎尖与‘D’叶样本分别于处理后1、2、3、4、5、6及8天的上午8:00至8:30间采集，并于当日中午前在实验室完成处理。设置叶基样本作为对照，以便与非茎尖组织进行比较，因为切除的茎尖上仍保留有叶原基。所有样本均保存于-80℃冰箱，直至在液氮中研磨成粉以进行RNA提取。采用Qiagen RNeasy植物总RNA提取迷你试剂盒（Qiagen，货号#74904），按照厂商说明书从茎尖与叶基中提取总RNA。使用DNA-free DNA去除试剂盒（Life Technologies，货号#AM1906M）去除基因组DNA。每个采样阶段设置3个生物学重复，每个重复包含3个茎尖或叶基样本。取2 µg总RNA，使用TruSeq链特异性mRNA LT建库试剂盒（Illumina，美国），按照厂商说明书构建mRNA测序文库。通过琼脂糖凝胶电泳评估RNA测序文库的片段大小（反应体系：1 μL样本 + 1 μL 6×上样缓冲液，1%琼脂糖凝胶，1×TBE缓冲液，60V电泳30分钟），并取1 μL样本使用Qubit荧光定量仪（Invitrogen，美国）搭配DNA HS检测试剂盒进行文库定量。将多重混合后的文库置于Illumina HiSeq4000测序平台进行测序，读长为50 nt。使用NEBNext多重小分子RNA建库试剂盒（适配Illumina平台，货号E7300，NEB，马萨诸塞州伊普斯维奇），按照厂商说明书从总RNA中构建小分子RNA测序文库。通过qPCR对文库进行定量，随后在测序泳道中进行测序，读长为50 nt。