Thiel Johannes, Weier Diana, Sreenivasulu Nese, Strickert Marc, Weichert Nicola, Melzer Michael, Czauderna Tobias, Wobus Ulrich, Weber Hans, Weschke Winfriede
Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung, D-06466 Gatersleben, Germany.
Plant Physiol. 2008 Nov;148(3):1436-52. doi: 10.1104/pp.108.127001. Epub 2008 Sep 10.
Nucellar projection (NP) and endosperm transfer cells (ETC) are essential tissues in growing barley (Hordeum vulgare) grains, responsible for nutrient transfer from maternal to filial tissues, endosperm/embryo nutrition, and grain development. A laser microdissection pressure catapulting-based transcriptome analysis was established to study NP and ETC separately using a barley 12K macroarray. A major challenge was to isolate high-quality mRNA from preembedded, fixed tissue while maintaining tissue integrity. We show that probes generated from fixed and embedded tissue sections represent largely the transcriptome (>70%) of nonchemically treated and nonamplified references. In NP, the top-down gradient of cellular differentiation is reflected by the expression of C3HC4-type ubiquitin ligases and different histone genes, cell wall biosynthesis and expansin/extensin genes, as well as genes involved in programmed cell death-related proteolysis coupled to nitrogen remobilization, indicating distinct areas simultaneously undergoing mitosis, cell elongation, and disintegration. Activated gene expression related to gibberellin synthesis and function suggests a regulatory role for gibberellins in establishment of the differentiation gradient. Up-regulation of plasmalemma-intrinsic protein and tonoplast-intrinsic protein genes indicates involvement in nutrient transfer and/or unloading. In ETC, AP2/EREBP-like transcription factors and ethylene functions are transcriptionally activated, a response possibly coupled to activated defense mechanisms. Transcriptional activation of nucleotide sugar metabolism may be attributed to ascorbate synthesis and/or cell wall biosynthesis. These processes are potentially controlled by trehalose-6-P synthase/phosphatase, as suggested by expression of their respective genes. Up-regulation of amino acid permeases in ETC indicates important roles in active nutrient uptake from the apoplastic space into the endosperm.
珠心突起（NP）和胚乳转移细胞（ETC）是大麦（Hordeum vulgare）籽粒生长过程中的重要组织，负责从母体组织向子代组织转运营养物质、胚乳/胚营养供应以及籽粒发育。我们建立了一种基于激光显微切割压力弹射技术的转录组分析方法，使用大麦12K宏阵列分别研究NP和ETC。一个主要挑战是从预先包埋、固定的组织中分离高质量的mRNA，同时保持组织完整性。我们发现，从固定和包埋的组织切片生成的探针在很大程度上代表了未经化学处理和未扩增的对照样本的转录组（>70%）。在NP中，C3HC4型泛素连接酶和不同组蛋白基因、细胞壁生物合成和扩展蛋白/伸展蛋白基因以及与程序性细胞死亡相关的蛋白水解和氮素再利用相关基因的表达反映了细胞分化的自上而下梯度，表明不同区域同时进行有丝分裂、细胞伸长和解体。与赤霉素合成和功能相关的基因表达激活表明赤霉素在分化梯度建立中起调节作用。质膜内在蛋白和液泡膜内在蛋白基因的上调表明其参与营养物质的转运和/或卸载。在ETC中，AP2/EREBP样转录因子和乙烯功能被转录激活，这种反应可能与激活的防御机制有关。核苷酸糖代谢的转录激活可能归因于抗坏血酸合成和/或细胞壁生物合成。这些过程可能受海藻糖-6-磷酸合酶/磷酸酶控制，其各自基因的表达表明了这一点。ETC中氨基酸通透酶的上调表明其在将质外体空间中的营养物质主动吸收到胚乳中起重要作用。
