Differences in bundle sheath and mesophyll cell development between C4 and C3 plants
在這個研究中我們建立了一套研究特定組織功能之系統，其中包含特定組織轉錄組之建立，生物訊息分析與鑑定，再利用病毒誘導基因沉默（VIGS）技術進行預測的調控因子初篩，最終利用CRISPR/Cas9基因編輯技術來做功能研究。首先我們建立冷凍切片系統，LCM 系統，low input RNA-seq library system。此系統可讓我們獲得高品質的RNA 跟轉錄組。我們總共擷取22組特定組織細胞包括(4組 BS cells; 3 組PM cells; 2 組M cells; 2組vascular cells)，每組兩重複。並完成18組轉錄組包括(4組 BS cells; 3組PM cells; 2 組M cells)， 每組兩重複。經由生物訊息的分析組間的差異，鑑定出組間表現差異的基因，更進一步篩選出調控花環結構或葉脈系統的發育上的基因。再利用病毒誘導基因沉默（VIGS）技術進行預測的調控因子之初篩，最終利用CRISPR/Cas9基因編輯技術來做功能研究。經由此系統之研究，鑑定出因基因功能喪失之植株影響維管束鞘細胞、葉肉細胞發育造成維管束的束型改變，進而影響葉片發育、生長遲緩、最終導致產量下降。
In this project we created a specific tissue transcriptome system in which we established a cryosection system, a LCM system, and a low input RNA-seq library system. Thess systems allow us to obtain high-quality RNA and transcriptome data. We collected 22 groups of tissue-specific cells, including 4 groups of BS cells, 3 groups of PM cells, 2 groups of M cells, and 2 groups of vascular cells. In addition, we completed 18 groups of transcriptiomes including 4 groups of BS cells, 3 groups of PM cells, and 2 groups of M cells, each group with two replicates. Bioinformatics analyses of the transcriptomes were conducted to identify genes differentially expressed between transcriptomes and especially transcription factor (TF) genes responsible for the differentiation in regulation between BS and M cells. Finally, through an approach by combining VIGS and CRISPR/Cas9 systems, knockout plants displaying a dramatic reduction in growth as well as disruptions in vascular patterning and spacing, leaf development, and BS and M cell patterning and development will be collected. In summary, this project used a combination of tissue specfic high-throughput technologies, bioinformatics skills, and molecular biology techniques to identify regulators involved in leaf and Kranz antomy development.
Figure 1. LCM-mediated isolation of Kranz tissue specific cells.
Sections before LCM (first column), sections after LCM (middle column), and captured cells on the cap (last column). (Notation: 4 stages of Kranz cells : 3, 4, 5, and 6 BS GM cells; 3 stages of palisade-like M cells (the same 3, 4, 5 BS cell stages PM cells); 2 stages of M cells (1 and 2 M cells); 2 stages of V cells (5 BS-V cells stage vascular cells; 10BS-Vcells stage vascular cells).
Figure 2 Whole-plant and leaf vascular phenotypes of knockout plants.
Photograph of 5-week-old plants. Knockout plants are smaller than W (wild-type), and have small grains (B) and thinner leaves (C). Light micrographs of Lugol’s staining leaf tissue (IKI-stained to reveal starch) shows that the regular parallel vein pattern (D-W) is disrupted in knockout leaves (D-2-8-7). Light microscope images of veins in knockout plant, a transverse section obtained two minor veins with merged bundle sheath (BS) cells (E).