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. 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）蛋白以串联的30至40个氨基酸序列基序为特征，是植物中的一个大型基因家族。现有研究表明，这类PPR蛋白在拟南芥和水稻的细胞器RNA代谢及植物生长发育过程中发挥重要作用。然而木本植物中PPR基因的功能仍大多未被阐明。本研究在杨树基因组中共鉴定并注释了626个携带PPR基序的PPR基因，并对杨树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、系统发育关系及基因复制事件。转录组分析显示，154个PtrPPR基因可被生物和非生物胁迫诱导，包括褐盘二孢（Marssonina brunnea）、水杨酸（salicylic acid, SA）、茉莉酸甲酯（methyl jasmonate, MeJA）、机械损伤、低温与盐胁迫。实时定量反转录PCR（quantitative Reverse Transcription PCR, qRT-PCR）分析进一步验证了11个PtrPPR基因在不同胁迫下的表达模式。本研究结果有助于更全面地解析PPR蛋白的功能，并为提升杨树抗逆性提供了新的研究思路。

研究目的：本研究旨在比较野生型与经过生物、非生物胁迫处理的毛果杨（Populus trichocarpa, P. trichocarpa）中PPR基因的表达谱，处理方式包括褐盘二孢侵染、SA处理、MeJA处理、机械损伤、低温及盐胁迫。

植物材料处理：
激素处理：将5 mM水杨酸（溶于去离子水）与1 mM茉莉酸甲酯（溶于0.1%体积分数乙醇）以5 mL液滴形式施于每株植株，处理后立即用透明盖子覆盖植株。
真菌接种：接种24小时后收集叶片。将3月龄植株的叶片接种褐盘二孢多芽专化型（M. brunnea f.sp. multigermtubi），将直径6 mm的菌丝块置于离体叶片上（每株至少取3片叶片），随后将叶片置于铺有湿润滤纸的培养皿中，在保湿培养箱内培养3天。
低温胁迫：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：使用无菌针刺破杨树幼叶后，将叶片置于20℃环境下放置2小时，随后收集样品。
盐胁迫：将4周龄的杨树苗置于添加100 mM氯化钠的全强度霍格兰德溶液（Hoagland’s solution）中处理2天。

野生型植株在温室中培养，培养条件为25℃、14小时光照/10小时黑暗。此外，所有胁迫处理、病原菌侵染、对照（CK）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。