Comparison of gene expression between cna-2 phb-13 phv-11, cna-2 phb-13 phv-11 rev-6, and wild type (Col er-2) in 0.5 mm root tips of Arabidopsis.. Arabidopsis thaliana

The class III HD-ZIP transcription factors regulate vascular patterning in Arabidopsis thaliana roots. In this expression study we compare the expression profile of the cna-2 phb-13 phv-11 and cna-2 phb-13 phv-11 rev-6 mutants to their wild type. The results are presented in PHABULOSA mediates an auxin signaling loop to regulate vascular patterning in Arabidopsis by Christina Joy Müller, Ana Elisa Valdés, Guodong Wang, Prashanth Ramachandran, Lisa Beste, Daniel Uddenberg, and Annelie Carlsbecker, accepted for publication in Plant Physiology Nov. 2015. Plant vascular tissues, xylem and phloem, differentiate in distinct patterns from procambial cells as an integral transport system for water, sugars and signaling molecules. Procambium formation is promoted by high auxin levels activating class III homeodomain leucine zipper (HD-ZIP III) transcription factors (TFs). In the root of Arabidopsis thaliana, HD-ZIP III TFs dose-dependently govern the patterning of the xylem axis, with higher levels promoting metaxylem cell identity in the central axis and lower levels protoxylem at its flanks. It is, however, unclear by what mechanisms the HD-ZIP III TFs control xylem axis patterning. Here we present data suggesting that an important mechanism is their ability to moderate auxin response. We found that changes in HD-ZIP III TF levels affect the expression of genes encoding core auxin response molecules. We show that one of the HD-ZIP III TFs, PHABULOSA, directly binds the promoter of both MONOPTEROS/AUXIN RESPONSE FACTOR5 (MP/ARF5), a key factor in vascular formation, and IAA20, encoding an AUX/IAA protein which is stable in the presence of auxin and able to interact with and repress MP activity. The double mutant of IAA20 and its closest homologue IAA30 forms ectopic protoxylem, while overexpression of IAA30 causes discontinuous protoxylem and occasional ectopic metaxylem, similar to a weak loss-of-function mp-mutant. Our results provide evidence that HD-ZIP III TFs directly affect auxin response and mediate a feed forward loop formed by MP and IAA20 that may focus and stabilize auxin response during vascular patterning and differentiation of xylem cell types. Overall design: Root tips of 4 days post germination Arabidopsis thaliana cna-2 phb-13 phv-11 and cna-2 phb-13 phv-11 rev-6 mutants and Col er-2 grown vertically were manually microdissected to ca 0.5 mm. The cna-2 phb-13 phv-11 rev-6 homozygous plants originated from a segregating cna-2 phb-13 phv-11 rev-6/+ population and were identified based on their radial cotyledon phenotype, that can be easily distinguished. Three biological replicates were included for each genotype. RNA was prepared from the root tips and hybridized on Affymetrix ATH1 microarrays.

III类同源域亮氨酸拉链转录因子（homeodomain leucine zipper, HD-ZIP III）调控拟南芥（Arabidopsis thaliana）根的维管模式建成。本表达谱研究比较了cna-2 phb-13 phv-11与cna-2 phb-13 phv-11 rev-6突变体与其野生型的表达特征。相关研究结果见于Christina Joy Müller、Ana Elisa Valdés、Guodong Wang、Prashanth Ramachandran、Lisa Beste、Daniel Uddenberg及Annelie Carlsbecker发表于2015年11月、已被《Plant Physiology（植物生理学）》接收的论文《PHABULOSA介导生长素信号环路调控拟南芥维管模式建成》。

植物维管组织（木质部与韧皮部）由原形成层细胞以特异性模式分化而来，是负责运输水分、糖分及信号分子的整合性转运系统。高生长素水平可激活III类同源域亮氨酸拉链（HD-ZIP III）转录因子，进而促进原形成层的形成。在拟南芥根中，HD-ZIP III转录因子以剂量依赖方式调控木质部轴的模式建成：较高水平的HD-ZIP III可促进中央轴的后生木质部细胞身份确立，较低水平则促进侧翼的原生木质部形成。然而，HD-ZIP III转录因子调控木质部轴模式建成的具体分子机制仍不明确。

本研究数据显示，其关键机制之一是HD-ZIP III转录因子能够调节生长素响应。我们发现HD-ZIP III转录因子水平的变化会影响核心生长素响应分子编码基因的表达。研究证实，HD-ZIP III转录因子家族成员PHABULOSA可直接结合维管形成关键因子MONOPTEROS/生长素响应因子5（MP/ARF5）的启动子，以及编码生长素响应蛋白IAA20的基因启动子；IAA20蛋白在生长素存在时保持稳定，能够结合并抑制MP的活性。IAA20与其最近同源基因IAA30的双突变体可形成异位原生木质部，而IAA30过表达会导致原生木质部不连续并偶发异位后生木质部，该表型与功能减弱的mp突变体相似。本研究结果证明，HD-ZIP III转录因子可直接影响生长素响应，并介导由MP和IAA20构成的前馈环路，该环路可能在维管模式建成及木质部细胞类型分化过程中聚焦并稳定生长素响应。

整体实验设计：将萌发后4天的拟南芥cna-2 phb-13 phv-11、cna-2 phb-13 phv-11 rev-6突变体及Col er-2野生型植株垂直培养，手动显微切割其根尖至约0.5mm长度。cna-2 phb-13 phv-11 rev-6纯合植株来自分离群体cna-2 phb-13 phv-11 rev-6/+，并通过易于辨识的子叶径向表型进行鉴定。每个基因型设置3个生物学重复。提取根尖总RNA，在Affymetrix ATH1微阵列（Affymetrix ATH1 microarrays）上进行杂交实验。