從C3光合作用途徑演化而來的C4光合作用，構成了一個複雜的綜合性狀，包含了葉部生化和結構性的改變。雖然目前已經很清楚C4植物的光合作用的生化機制，但是C4植物葉子結構改變的機制仍然存在許多疑問。在C3植物演化成C4植物的過程中，葉脈密度增加是C4植物的特殊結構特徵，被認為是C3植物演化成C4植物過程中的關鍵步驟，也是未來將C3作物改變成C4作物的重要關鍵。我的假說是:在C4植物葉子的發育過程中，由於生長素的合成與運輸被提高了而促使葉脈密度增加。為了檢測這個假設，我進行了下列的研究。首先，我從同屬於白花菜科(Cleomaceae)的C3醉蝶花(Tarenaya hassleriana)和C4白花菜(Gynandropsis gynandra)葉子發育的早期，每一品種各取得了兩個轉錄體(transcriptomes)。我利用了RNA-seq的技術，比較了在這兩種白花菜科植物的基因表現的差異後，發現在C4白花菜裡，多數與生長素(auxin)合成途徑或負責輸送生長素有關基因的表現量提高，而且降低了色胺酸(生長素的上游前驅物)合成的抑制轉錄因子MYC2的表現量。第二，我發現白花菜發育的葉子裡，具有較高量的生長素；同樣的，在玉米的葉子原基組織(發育成C4光合作用組織)和玉米外殼原基組織(發育成C3光合作用組織)的比較也發現同樣的結果。第三，我發現當C3植物阿拉伯芥myc2發生突變而失去功能時，會造成生長素和植物葉脈密度的提升。第四，利用生長素合成抑制劑，yucasin，來處理白花菜會降低葉子的葉脈密度。第五，利用生長素運送抑制劑，NPA，來處理白花菜和醉蝶花則會減少新生植物葉片高層級葉脈的數目。最後，當負責輸送生長素的基因pin1或lax2發生突變時，會降低葉脈的密度。這些的研究結果指出生長素的合成基因和生長素輸送基因在控制葉脈密度形成上扮演重要的角色。因此我推斷出演化過程中提高生長素的合成和輸送是造成C4植物葉子形成高密度葉脈的分子機制。

The complex syndromes of C4 photosynthesis include several biochemical and anatomical modifications evolved from the ancestral C3 photosynthetic pathway. Although the biochemical modifications in C4 plants are well understood, the mechanisms responsible for C4 anatomical modifications are still largely unknown. High vein density, which is a distinctive anatomical trait of C4 leaves, is central to C3-to-C4 evolution and is considered a key step in the conversion of C3 to C4-like crops in the future. I tested the hypothesis that high vein density in C4 leaves is due to elevated auxin biosynthesis and transport in developing leaves. To test this hypothesis I conducted the following studies. First, I obtained transcriptomes of developing leaves from Tarenaya hassleriana and Gynandropsis gynandra, a C3 and a C4 species in Cleomaceae. I analyzed and compared expressed genes of the two Cleomaceae species by RNA-seq. In C4 G. gynandra many genes promoting auxin biosynthesis or transport were up-regulated while the gene for MYC2, a transcription factor that suppresses the biosynthesis of tryptophan, which is a precursor of auxin, was down-regulated. Second, I found higher auxin contents in the developing leaves of G. gynandra. The same observation held for maize foliar (C4) and husk (C3) leaf primordia. Third, I found increased auxin contents and vein densities in both of the two Arabidopsis thaliana loss-of-function myc2 mutants. Fourth, treating G. gynandra with yucasin, an auxin biosynthesis inhibitor, reduced leaf vein density. Fifth, treating plants with NPA, an auxin transport inhibitor, led to much fewer higher-order (minor) veins in new leaves. Finally, both A. thaliana auxin efflux transporter pin1 and influx transporter lax2 mutants showed reduced vein numbers. These observations point to the central role of auxin biosynthesis and transport in vein density control. I conclude that elevated biosynthesis and transport of auxin is the molecular basis underlying high vein density in C4 leaves.

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