Table_1_Overexpression of the Soybean NAC Gene GmNAC109 Increases Lateral Root Formation and Abiotic Stress Tolerance in Transgenic Arabidopsis Plants.docx

NACs are plant-specific transcription factors that have crucial roles in plant development and biotic and/or abiotic stress responses. This study characterized the functions of the soybean NAC gene GmNAC109 using an overexpression construct in Arabidopsis lines. Sequence analysis revealed that GmNAC109 is highly homologous to ATAF1 (Arabidopsis Transcription Activation Factor 1), which regulates biotic and abiotic stress responses. GmNAC109 protein localized to the nucleus and its C-terminal domain exhibited transcriptional activation activity. Salt, dehydration, and cold stresses significantly increased expression of GmNAC109 in soybean. Similarly, Arabidopsis plants overexpressing GmNAC109 were more tolerant to drought and salt stress than wild-type Col-0 plants. Stress response-related genes, such as DREB1A (drought-responsive element-binding 1A), DREB2A, AREB1 (ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN 1), AREB2, RD29A (RESPONSIVE TO Desiccation 29A), and COR15A (COLD REGULATED 15A) were upregulated in GmNAC109-overexpressing transgenic Arabidopsis lines. The transgenic lines showed upregulation of the ABA-responsive genes ABI1 (ABA INSENSITIVE 1) and ABI5 and hypersensitivity to ABA. However, GmNAC109 did not increase expression of the ABA-biosynthetic gene NCED3 (NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3) and endogenous ABA content in the transgenic lines. Overexpression of GmNAC109 significantly increased lateral root formation in transgenic Arabidopsis lines. Expression of AIR3 (AUXIN-INDUCED IN ROOT CULTURES 3) and ARF2 (AUXIN RESPONSE FACTOR 2) was increased and decreased in these transgenic lines, respectively, indicating that GmNAC109 is involved in the auxin signaling pathway and thereby helps to regulate hairy root formation. Our results provide a basis for development of soybean lines with improved tolerance to abiotic stresses via genetic manipulation.

NAC家族转录因子是植物特有的转录因子（transcription factors），在植物生长发育以及生物和/或非生物胁迫响应中发挥关键作用。本研究通过在拟南芥（Arabidopsis）株系中构建过表达载体，对大豆NAC基因GmNAC109的功能进行了系统鉴定与分析。序列分析结果显示，GmNAC109与调控生物及非生物胁迫响应的ATAF1（Arabidopsis Transcription Activation Factor 1，拟南芥转录激活因子1）具有高度同源性。GmNAC109蛋白定位于细胞核，其C端结构域具备转录激活活性。盐胁迫、脱水胁迫与低温胁迫均可显著诱导大豆中GmNAC109基因的表达。同样地，过表达GmNAC109的拟南芥植株相较于野生型Col-0株系，对干旱与盐胁迫的耐受性显著增强。在过表达GmNAC109的转基因拟南芥株系中，诸多胁迫响应相关基因的表达均被上调，包括DREB1A（drought-responsive element-binding 1A，干旱应答元件结合蛋白1A）、DREB2A、AREB1（ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN 1，脱落酸应答元件结合蛋白1）、AREB2、RD29A（RESPONSIVE TO Desiccation 29A，脱水应答蛋白29A）以及COR15A（COLD REGULATED 15A，低温调控蛋白15A）。该转基因株系中脱落酸（ABA）应答基因ABI1（ABA INSENSITIVE 1，ABA不敏感1）与ABI5的表达同样被上调，且对脱落酸（ABA）表现出超敏感性。但GmNAC109并未提升脱落酸（ABA）生物合成基因NCED3（NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3，顺式环氧类胡萝卜素双加氧酶3）的表达水平，转基因株系的内源脱落酸（ABA）含量亦未发生改变。过表达GmNAC109可显著促进转基因拟南芥株系的侧根生成。在此类转基因株系中，AIR3（AUXIN-INDUCED IN ROOT CULTURES 3，根系培养中生长素诱导基因3）的表达被上调，而ARF2（AUXIN RESPONSE FACTOR 2，生长素响应因子2）的表达则被下调，这表明GmNAC109参与生长素信号通路，进而参与调控毛状根的形成。本研究结果为通过基因工程手段培育抗非生物胁迫能力提升的大豆品种提供了重要的理论依据。