Genome-wide investigation of pentatricopeptide repeat (PPR) gene family in poplar and their expression analysis in response to biotic and abiotic stresses

Pentatricopeptide repeat (PPR) proteins, which are characterized by tandem 30-40 amino acid sequence motifs, constitute a large gene family in plants. These known PPR proteins have been identified to play important roles in organellar RNA metabolism and plant development in Arabidopsis and rice. However, functions of PPR genes in woody species remain still largely unknown. Here, we identified and characterized a total of 626 PPR genes containing PPR motifs in the poplar genome. A comprehensive genome-wide analysis of the poplar PPR gene family was performed, including chromosomal location, phylogenetic relationships, gene duplication. Transcriptomic analyses identified that 154 of the PtrPPR genes were induced by biotic and abiotic treatments, including Marssonina brunnea, salicylic acid (SA), methyl jasmonate (MeJA), wounding, cold and salinity. Quantitative RT-PCR analysis further confirmed the expression profiles of 11 PtrPPR genes under different stresses. Our results contribute to a more comprehensive understanding the roles of PPR proteins and provided an insight for improving the stress tolerance in poplar. Overall design: Purpose: The goal of this study is to compare the PPR genes profile in Wild Type with biotic and abiotic treatment of P. trichocarpa. Treatment including M. brunnea, SA and MeJA, wound, low temperature, and salinity. Plant material treatment: Hormone treatments: SA (5 mM in water) and MeJA [1mM in 0.1% (v/v) ethanol] were applied at the different concentrations as 5 ml droplets on each plant. The treated plants were immediately covered with a transparent lid. Fungal inoculation: The leaves were collected after 24 h. Leaves of three-month-old plants were inoculated with M. brunnea f.sp. multigermtubi. Mycelial plugs (6 mm) were placed on excised leaves (at least three leaves for each plant). These leaves were incubated in Petri dishes with humid filter paper in a humid chamber for 3 d. Low temperature stress: The healthy, well-hydrated plants were transferred to a growth chamber at 4°C under the same light and photoperiodic conditions for 1 h. After cold treatment, plants were allowed to recover at 20°C for 1 h. Wounding stress: For the wounding treatment, the young leaves of poplar plants were harvested after being punctured with sterile needles and placed at 20°C for 2 h. Salinity stress: The four-week-old seedlings were subjected to salt stress. Saline treatments had the NaCl concentrations of 100 mM added to full-strength Hoagland's solution for 2 d. Wild type plant were grown in a greenhouse at 25°C under a 14/10 h light/dark cycle. Additionally, the leaves applied for all stress treatments, pathogen infection, CK and DGE analysis were excised from the second and third internodes.

五肽重复序列（Pentatricopeptide repeat, PPR）蛋白以串联排布的30~40个氨基酸序列基序为典型特征，是植物中一个庞大的基因家族。目前已在拟南芥和水稻中鉴定出多种PPR蛋白，它们在细胞器RNA代谢与植物生长发育过程中发挥重要调控作用。然而，木本植物中PPR基因的功能仍在很大程度上未被阐明。本研究在杨树基因组中共鉴定出626个携带PPR基序的PPR基因，并对其进行了系统表征。我们对杨树PPR基因家族开展了全基因组范围的综合分析，涵盖染色体定位、系统发育关系以及基因复制事件。转录组分析结果显示，共有154个毛果杨PtrPPR基因受生物与非生物胁迫诱导，这些胁迫包括杨褐盘二孢菌（Marssonina brunnea）侵染、水杨酸（SA）处理、茉莉酸甲酯（MeJA）处理、机械损伤、低温以及盐胁迫。实时定量逆转录PCR（qRT-PCR）分析进一步验证了11个PtrPPR基因在不同胁迫条件下的表达模式。本研究结果有助于更全面地解析PPR蛋白的生物学功能，同时为提升杨树的胁迫耐受能力提供了理论依据与研究思路。

整体实验设计：
研究目的：本研究旨在对比分析毛果杨（P. trichocarpa）野生型与经生物、非生物胁迫处理后的PPR基因表达谱差异。所涉及的胁迫处理包括杨褐盘二孢菌侵染、SA处理、MeJA处理、机械损伤、低温胁迫以及盐胁迫。

植物材料处理：
激素处理：将浓度为5 mM的水杨酸水溶液、以及溶于0.1%（体积分数）乙醇的1 mM茉莉酸甲酯溶液，以每株5 mL的滴加量施用于植株，处理后立即用透明盖子覆盖植株。
真菌接种处理：将3月龄植株的叶片剪下，用杨褐盘二孢菌多芽变种（M. brunnea f.sp. multigermtubi）的菌丝块（直径6 mm）进行接种，每株至少接种3片叶片。接种后的叶片置于铺有湿润滤纸的培养皿中，于加湿培养箱内培养3天，样本于处理24小时后采集。
低温胁迫处理：将生长健壮、水分充足的植株转移至光照与光周期条件一致的4℃生长箱中处理1小时，低温处理结束后，将植株置于20℃环境下恢复1小时。
机械损伤胁迫处理：用无菌针刺伤杨树幼叶，随后将叶片置于20℃环境下放置2小时，之后采集样本。
盐胁迫处理：将生长4周的杨树苗置于添加了100 mM NaCl的完全霍格兰德营养液中培养2天，以进行盐胁迫处理。
野生型植株于温室中培养，培养条件为25℃、14小时光照/10小时黑暗的光周期。
此外，所有胁迫处理、病原菌侵染处理、对照组（CK）以及数字基因表达谱（DGE）分析所用的叶片，均取自植株的第2和第3节间部位。