Two Subclasses of Differentially Expressed TPS1 Genes and Biochemically Active TPS1 Proteins May Contribute to Sugar Signalling in Kiwifruit Actinidia chinensis

Trehalose metabolism and its intermediate trehalose-6-phosphate (T6P) are implicated in sensing and signalling sucrose availability. Four class I TREHALOSE-6-PHOSPHATE SYNTHASE (TPS1) genes were identified in kiwifruit, three of which have both the TPS and trehalose-6-phosphate phosphatase (TPP) domain, while the fourth gene gives rise to a truncated transcript. The transcript with highest sequence homology to Arabidopsis TPS1, designated TPS1.1a was the most highly abundant TPS1 transcript in all examined kiwifruit tissues. An additional exon giving rise to a small N-terminal extension was found for two of the TPS1 transcripts, designated TPS1.2a and TPS1.2b. Homology in sequence and gene structure with TPS1 genes from Solanaceae suggests they belong to a separate, asterid-specific class I TPS subclade. Expression of full-length and potential splice variants of these two kiwifruit TPS1.2 transcripts was sufficient to substitute for the lack of functional TPS1 in the yeast tps1Δ tps2Δ mutant, but only weak complementation was detected in the yeast tps1Δ mutant, and no or very weak complementation was obtained with the TPS1.1a construct. Transgenic Arabidopsis lines expressing kiwifruit TPS1.2 under the control of 35S promoter exhibited growth and morphological defects. We investigated the responses of plants to elevated kiwifruit TPS1 activity at the transcriptional level, using transient expression of TPS1.2a in Nicotiana benthamiana leaves, followed by RNA-seq. Differentially expressed genes were identified as candidates for future functional analyses.

海藻糖代谢及其中间产物海藻糖-6-磷酸（trehalose-6-phosphate, T6P）参与蔗糖可利用性的感知与信号传导过程。研究团队在猕猴桃中鉴定出4个I类海藻糖-6-磷酸合酶（TPS1）基因，其中3个同时包含TPS和海藻糖-6-磷酸磷酸酶（trehalose-6-phosphate phosphatase, TPP）结构域，第4个基因则产生截短转录本。与拟南芥（Arabidopsis）TPS1序列同源性最高的转录本被命名为TPS1.1a，其为所有检测过的猕猴桃组织中丰度最高的TPS1转录本。针对两个TPS1转录本——TPS1.2a与TPS1.2b，研究发现其存在一段额外外显子，可编码一段短小的N端延伸序列。该类基因与茄科（Solanaceae）TPS1基因在序列及基因结构上的同源性提示，它们隶属于一个独立的、菊类植物特异性的I类TPS亚簇。将这两个猕猴桃TPS1.2转录本的全长序列及潜在剪接变体进行异源表达，可互补酵母tps1Δ tps2Δ突变体中功能性TPS1的缺失，但在酵母tps1Δ突变体中仅能观测到较弱的互补效应；而利用TPS1.1a构建体进行实验时，则未检测到或仅能观测到极微弱的互补作用。在35S启动子（35S promoter）调控下表达猕猴桃TPS1.2的转基因拟南芥株系，表现出生长与形态学异常表型。为从转录组层面探究植物对升高的猕猴桃TPS1活性的响应，本研究将TPS1.2a瞬时转化本氏烟草（Nicotiana benthamiana）叶片，随后开展RNA测序（RNA-seq）分析。最终筛选得到的差异表达基因，可作为后续功能研究的候选靶点。