Department of Chemical Engineering, Northeastern University, Boston, MA, 02115, USA.
Department of Bioengineering, Northeastern University, Boston, USA.
Plant Cell Rep. 2024 May 14;43(6):141. doi: 10.1007/s00299-024-03208-9.
A GLK homologue was identified and functionally characterized in Catharanthus roseus. Silencing CrGLK with VIGS or the chloroplast retrograde signaling inducer lincomycin increased terpenoid indole alkaloid biosynthesis. Catharanthus roseus is the sole source of the chemotherapeutic terpenoid indole alkaloids (TIAs) vinblastine and vincristine. TIA pathway genes, particularly genes in the vindoline pathway, are expressed at higher levels in immature versus mature leaves, but the molecular mechanisms responsible for this developmental regulation are unknown. We investigated the role of GOLDEN2-LIKE (GLK) transcription factors in contributing to this ontogenetic regulation since GLKs are active in seedlings upon light exposure and in the leaf's early development, but their activity is repressed as leaves age and senesce. We identified a GLK homologue in C. roseus and functionally characterized its role in regulating TIA biosynthesis, with a focus on the vindoline pathway, by transiently reducing its expression through two separate methods: virus-induced gene silencing (VIGS) and application of chloroplast retrograde signaling inducers, norflurazon and lincomycin. Reducing CrGLK levels with each method reduced chlorophyll accumulation and the expression of the light harvesting complex subunit (LHCB2.2), confirming its functional homology with GLKs in other plant species. In contrast, reducing CrGLK via VIGS or lincomycin increased TIA accumulation and TIA pathway gene expression, suggesting that CrGLK may repress TIA biosynthesis. However, norflurazon had no effect on TIA gene expression, indicating that reducing CrGLK alone is not sufficient to induce TIA biosynthesis. Future work is needed to clarify the specific molecular mechanisms leading to increased TIA biosynthesis with CrGLK silencing. This is the first identification and characterization of GLK in C. roseus and the first investigation of how chloroplast retrograde signaling might regulate TIA biosynthesis.
在长春花中鉴定并功能表征了一个 GLK 同源物。通过 VIGS 或质体逆行信号诱导物林可霉素沉默 CrGLK 会增加萜烯吲哚生物碱生物合成。长春花是化学治疗性萜烯吲哚生物碱（TIAs）长春碱和长春新碱的唯一来源。TIAs 途径基因，特别是长春质碱途径中的基因，在未成熟叶片中比成熟叶片表达水平更高，但负责这种发育调控的分子机制尚不清楚。我们研究了 GLK 转录因子在促成这种个体发育调控中的作用，因为 GLK 在光照下的幼苗和叶片早期发育中活跃，但随着叶片衰老，其活性受到抑制。我们在长春花中鉴定了一个 GLK 同源物，并通过两种独立的方法（病毒诱导的基因沉默（VIGS）和应用质体逆行信号诱导物，如 norflurazon 和林可霉素）暂时降低其表达，从而对其在调节 TIAs 生物合成中的作用进行了功能表征，重点是长春质碱途径。通过每种方法降低 CrGLK 水平都会减少叶绿素积累和光捕获复合体亚基（LHCB2.2）的表达，这证实了其与其他植物物种中的 GLK 具有功能同源性。相比之下，通过 VIGS 或林可霉素降低 CrGLK 水平会增加 TIAs 积累和 TIAs 途径基因表达，表明 CrGLK 可能抑制 TIAs 生物合成。然而，norflurazon 对 TIAs 基因表达没有影响，这表明单独降低 CrGLK 不足以诱导 TIAs 生物合成。需要进一步的工作来阐明导致 CrGLK 沉默时 TIAs 生物合成增加的具体分子机制。这是在长春花中鉴定和表征 GLK 的首次尝试，也是首次研究质体逆行信号如何调节 TIAs 生物合成。
