RNA-Sequencing Analysis of Abscission-related Transcriptome in Citrus Calyx Abscission Zone Reveals Distinguishing Profile of Phytohormone Expression Associated with Abscission of HLB-affected Sweet Orange

Citrus greening or huanglongbing (HLB) disease is caused by Candidatus liberibacter asiaticus (CLas) and associated with an increase in pre-harvest fruit drop, for which the molecular mechanisms remain unknown. In order to understand the molecular basis of the HLB-associated fruit abscission, by means of RNA-Sequencing analysis (RNA-Seq), transcriptomes in citrus calyx abscission zones were analyzed and compared among fruit dropped (D) or retained (R) from healthy (h) or HLB-diseased (d) trees upon shaking the trees. Cluster analysis based on the transcript reads indicates that dropped fruit from HLB-diseased trees (Dd) have largest distances from all other groups. Differentially expressed genes (DEGs) were identified between Dd and Rd, Dh and Rh, Rd and Rh, Dd and Dh. Wilcoxon test of the whole dataset of DEGs revealed that consistently up-regulated genes in Dd versus Rd and Dd versus Dh are in the functional categories of “secondary metabolism, lipid metabolism and hormone-ethylene and –jasmonate”; while those down-regulated genes didn’t show clear pattern of regulation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that the significant biological processes or pathways involved in HLB-related fruit abscission were those related to defense response, secondary metabolism and hormone signaling. Among them, “response to chitin” was the most significant (p= 9.95E-13) biological process, and “jasmonic acid biosynthesis” was the most significant (p= 4.6E-5) pathway. Genes related to synthesis and signaling of ethylene (ET) and jasmonate (JA) were consistently up-regulated, while abscisic acid, auxin, brassinosteroid, cytokinin, and gibberellin were generally down-regulated in Dd; but not in Dh. Consistent with the transcriptomic data, fruit ethylene production was detected in two third of the Dd fruit, but none of Rd, Dh or Rh fruit. And, in agreement with the hormone expression profiles, substantial numbers of downstream JA/ET-responsive defense related genes were up-regulated in Dd, but not in Dh. Thirty representative DEGs covering categories of hormone, secondary metabolism, and JA/ET responsive defense responses were verified by qRT-PCR. The results indicate that HLB-associated pre-harvest fruit abscission is mediated by JA/ET signaling, which has been known to be triggered by infection of necrotrophic pathogens. Overall design: Six-year old ‘Hamlin’ orange trees (Citrus sinensis (L.) Osbeck), about 2.5-3.0 m tall, on 'Swingle' citrumelo (C. paradisi Macf. × Poncirus trifoliata (L) Raf.) rootstock, in a commercial grove located in Southern Florida. Thirty six trees were selected for the experiment, of which eighteen were CLas negative (healthy) and the other eighteen were CLas positive (HLB), as tested by qPCR using the method of Li et al. (2006). The selected trees were similar in size, and all were grown under similar agro-climatic conditions and received common cultural practices and the grower’s standard pest and disease management. Fruit were harvested on 1 Dec. 2014 (during commercial harvest season). The ground under the trees was cleaned just before shaking the trees, and trees were shaken manually. For HLB-diseased trees (d), many of the fruit dropped (Dd) upon shaking the trees. For healthy trees (h), the forces of shaking were strong enough to get enough number of dropped fruit (Dh) from the trees. The dropped fruit (Dd or Dh) were collected, and the retained fruit on HLB-diseased (Rd) or healthy trees (Rh) were hand-picked off the trees (Fig. 1A). The fruit from nine trees were pooled together as a group. There were two groups (biological replicates) for each of the Dd, Rd, Dh and Rh. Thirty fruit were randomly picked from each of the Dd, Rd, Dh and Rh groups, and the AZ-C were excised (Fig.1B) and immediately frozen in liquid nitrogen and stored at –80 ºC for RNA isolation. Another thirty fruit from each of the groups were transported to the laboratory where fruit ethylene production was measured.

柚类黄龙病，亦称青果病（HLB），由亚洲类伯里茨氏菌（CLas）引起，并伴随收获前果实脱落率增加，其分子机制尚不明确。为探究与HLB相关的果实脱落分子基础，通过RNA测序分析（RNA-Seq），对柚花蕾脱落区转录组进行了分析，并比较了来自健康（h）或HLB病树（d）的脱落（D）或保留（R）果实。基于转录读数的聚类分析表明，HLB病树脱落果实（Dd）与所有其他组别之间的距离最大。在Dd与Rd、Dh与Rh、Rd与Rh、Dd与Dh之间鉴定出差异表达基因（DEGs）。Wilcoxon检验整个DEGs数据集显示，与Rd和Dh相比，Dd中持续上调的基因功能类别为“次生代谢、脂质代谢及激素乙烯和茉莉酸甲酯”；而下调基因未呈现明显的调控模式。基因本体（GO）和京都基因与基因组百科全书（KEGG）通路富集分析表明，参与HLB相关果实脱落的显著生物学过程或通路涉及防御反应、次生代谢和激素信号转导。其中，“响应几丁质”是最显著的生物学过程（p=9.95E-13），而“茉莉酸生物合成”是最显著的通路（p=4.6E-5）。与乙烯（ET）和茉莉酸甲酯（JA）的合成和信号转导相关的基因在Dd中持续上调，而脱落酸、生长素、 brassinosteroid、细胞分裂素和赤霉素在Dd中普遍下调；但在Dh中并非如此。与转录组数据一致，约三分之二Dd果实检测到乙烯产生，而Rd、Dh或Rh果实中均未检测到。与激素表达谱一致，大量下游JA/ET响应的防御相关基因在Dd中上调，但在Dh中未上调。三十个代表性DEGs，涵盖激素、次生代谢和JA/ET响应的防御反应类别，通过qRT-PCR进行验证。结果显示，HLB相关的收获前果实脱落由JA/ET信号传导介导，已知此信号传导由坏死性病原体感染触发。总体设计：选取六龄‘Hamlin’橙树（Citrus sinensis (L.) Osbeck），约2.5-3.0米高，种植于'Swingle' citrumelo（C. paradisi Macf. × Poncirus trifoliata (L) Raf.）砧木上，位于美国佛罗里达州南部的商业果园中。选取三十六棵树进行实验，其中十八棵为CLas阴性（健康），另十八棵为CLas阳性（HLB），通过qPCR采用Li等（2006）的方法进行检测。所选树木大小相似，均在相似的农业气候条件下生长，接受共同的文化管理和种植者的标准病虫害管理。果实于2014年12月1日（商业收获季节）收获。在摇树之前清洁树木下的地面，手动摇树。对于HLB病树（d），许多果实（Dd）在摇树时脱落。对于健康树（h），摇动的力量足够强，可以从树上获得足够的脱落果实（Dh）。收集脱落果实（Dd或Dh），从HLB病树（Rd）或健康树（Rh）上手工采摘保留的果实（图1A）。从九棵树中收集的果实作为一个组别。每个Dd、Rd、Dh和Rh组别有两个组（生物重复）。从每个Dd、Rd、Dh和Rh组别中随机挑选三十个果实，切取AZ-C部位（图1B），并立即在液氮中冷冻，存储于-80 ºC用于RNA提取。每组别另三十个果实被运送到实验室，测量果实乙烯产生。