Alternative splicing and gene duplication differentially shaped the regulation of isochorismate synthase in Populus and Arabidopsis

Isochorismate synthase (ICS) converts chorismate to isochorismate
for the biosynthesis of phylloquinone, an essential cofactor for
photosynthetic electron transport. ICS is also required for salicylic
acid (SA) synthesis during Arabidopsis defense. In several other
species, including Populus, SA is derived primarily from the phenylpropanoid
pathway. We therefore sought to investigate ICS
regulation in Populus to learn the extent of ICS involvement in SA
synthesis and defense. Arabidopsis harbors duplicated AtICS genes
that differ in their exon-intron structure, basal expression, and
stress inducibility. In contrast, we found a single ICS gene in
Populus and six other sequenced plant genomes, pointing to the
AtICS duplication as a lineage-specific event. The Populus ICS
encodes a functional plastidic enzyme, and was not responsive to
stresses that stimulated phenylpropanoid accumulation. Populus
ICS underwent extensive alternative splicing that was rare for the
duplicated AtICSs. Sequencing of 184 RT-PCR Populus clones revealed
37 alternative splice variants, with normal transcripts representing
_50% of the population. When expressed in Arabidopsis,
Populus ICS again underwent alternative splicing, but did not
produce normal transcripts to complement AtICS1 function. The
splice-site sequences of Populus ICS are unusual, suggesting a
causal link between junction sequence, alternative splicing, and ICS
function. We propose that gene duplication and alternative splicing
of ICS evolved independently in Arabidopsis and Populus in
accordance with their distinct defense strategies. AtICS1 represents
a divergent isoform for inducible SA synthesis during defense.
Populus ICS primarily functions in phylloquinone biosynthesis,
a process that can be sustained at low ICS transcript levels.

异分支酸合酶（Isochorismate synthase, ICS）可将分支酸转化为异分支酸，用于叶绿醌的生物合成——叶绿醌是光合电子传递过程中不可或缺的辅因子。ICS同时也是拟南芥（Arabidopsis）防御反应过程中水杨酸（salicylic acid, SA）合成所必需的。在包括杨属（Populus）在内的其他多个物种中，水杨酸主要通过苯丙烷途径合成。因此，我们旨在研究杨属中ICS的调控机制，以明确ICS在水杨酸合成与防御反应中的参与程度。
拟南芥拥有重复的AtICS基因，这些基因在外显子-内含子结构、基础表达水平以及胁迫诱导性上均存在差异。与之相反，我们在杨属及其他6个已测序植物基因组中仅发现单个ICS基因，这表明AtICS基因重复属于谱系特异性事件。
杨属ICS编码一种具有功能的质体酶，且不会响应可促进苯丙烷类物质积累的胁迫刺激。杨属ICS存在广泛的可变剪接现象，而重复的AtICS基因则极少发生该过程。对184个杨属逆转录聚合酶链式反应（RT-PCR）克隆的测序结果显示，其共存在37种可变剪接变体，正常转录本仅占总群体的约50%。
当在拟南芥中表达杨属ICS时，该基因同样会发生可变剪接，但无法产生正常转录本以互补AtICS1的功能。杨属ICS的剪接位点序列较为特殊，这提示剪接位点序列、可变剪接与ICS功能之间存在因果关联。
我们提出，ICS的基因重复与可变剪接在拟南芥与杨属中独立演化，以适配二者截然不同的防御策略。AtICS1代表了一种在防御反应中参与诱导型水杨酸合成的分化型同工型。杨属ICS的主要功能为叶绿醌生物合成，这一过程可在较低的ICS转录本水平下维持。