Establishment of a transgenic hairy root system in wild and domesticated watermelon (Citrullus lanatus) for studying root vigor under drought

Root vigor is an important trait for the growth of terrestrial plants, especially in water-deficit environments. Although deserts plants are known for their highly developed root architecture, the molecular mechanism responsible for this trait has not been determined. Here we established an efficient protocol for the genetic manipulation of two varieties of watermelon plants: a desert-grown wild watermelon that shows vigorous root growth under drought, and a domesticated cultivar showing retardation of root growth under drought stress. Agrobacterium rhizogenes-mediated transgenic hairy roots were efficiently induced and selected from the hypocotyls of these plants. Transgenic GUS expression was detected in the roots by RT-PCR and histochemical GUS staining. Moreover, a liquid culture system for evaluating their root growth was also established. Interestingly, growth of the hairy roots derived from domesticated variety of watermelon strongly inhibited under high osmotic condition, whereas the hairy roots derived from wild variety of watermelon retained substantial growth rates under the stress condition. The new protocol presented here offers a powerful tool for the comparative study of the molecular mechanism underlying drought-induced root growth in desert plants.     

干旱对杉木幼苗根系构型及非结构性碳水化合物的影响

通过比较不同时期不同强度干旱胁迫下杉木1年生盆栽苗地上部分生长、根系构型以及根系中非结构性碳水化合物含量（TNC）的变化,并分析各指标之间的相关性,探究杉木根系在干旱胁迫下的适应性策略以及抗旱生理机制,以期为杉木造林生产和水分管理提供科学依据和技术指导。结果表明：随着干旱程度的加强,杉木幼苗地上部分干重（SDW）、根干重（RDW）、根长（RL）、根表面积（SA）、根体积（RV）、根尖数（RT）、根系分支角度（Angle）、分形维数（FD）逐渐减小,根冠比（R/T）逐渐增大,根系拓扑指数（TI）、根系平均直径（RD）先增大后减小,比根长（SRL）先减小后增大。而根系连接长度（LL）、TNC、糖淀比在不同时期表现出不同的趋势。连接长度随着干旱胁迫的加强在30 d和60 d时表现出逐渐增加趋势而在90 d时则表现出先减小后增大的趋势。TNC在30 d和60 d时先增大后减小,但90 d时,呈逐渐下降的趋势。糖淀比随着干旱胁迫的加强在30 d和60 d时表现出先增加后减小趋势,90 d时,表现为先减小后增大。干旱胁迫显著影响根系在不同径级的分布长度,且随着胁迫时间的延长不断变化。杉木地上部分生长与根系生长指标（RL、SA、RV、RT、RDW）以及根系构型指标（Angle、FD）之间存在显著的正相关（P < 0.01）,根系平均直径与TNC存在显著的正相关（P < 0.05）。总之,杉木通过增加根系光合产物的积累、提高根系建成成本,增加有限成本下根系的复杂程度和延伸范围,降低根系分支角度,使根系"更陡更深"来适应不同强度的干旱胁迫。     

Programmed proteome response for drought avoidance/tolerance in the root of a C(3) xerophyte (wild watermelon) under water deficits

Water availability is a critical determinant for the growth and ecological distribution of terrestrial plants. Although some xerophytes are unique regarding their highly developed root architecture and the successful adaptation to arid environments, virtually nothing is known about the molecular mechanisms underlying this adaptation. Here, we report physiological and molecular responses of wild watermelon (Citrullus lanatus sp.), which exhibits extraordinarily high drought resistance. At the early stage of drought stress, root development of wild watermelon was significantly enhanced compared with that of the irrigated plants, indicating the activation of a drought avoidance mechanism for absorbing water from deep soil layers. Consistent with this observation, comparative proteome analysis revealed that many proteins induced in the early stage of drought stress are involved in root morphogenesis and carbon/nitrogen metabolism, which may contribute to the drought avoidance via the enhancement of root growth. On the other hand, lignin synthesis-related proteins and molecular chaperones, which may function in the enhancement of physical desiccation tolerance and maintenance of protein integrity, respectively, were induced mostly at the later stage of drought stress. Our findings suggest that this xerophyte switches survival strategies from drought avoidance to drought tolerance during the progression of drought stress, by regulating its root proteome in a temporally programmed manner. This study provides new insights into the complex molecular networks within plant roots involved in the adaptation to adverse environments.     

干旱胁迫对蒙古黄芪生长及根部次生代谢物含量的影响

以山西道地药材黄芪一年生幼苗为试验材料,设置常规水分条件(CK)、轻度干旱胁迫(A1)、中度干旱胁迫(A2)、重度干旱胁迫(A3)4个不同处理,研究土壤干旱胁迫对黄芪生长及生理的影响。结果表明:黄芪茎叶快速生长集中在出苗后80—120d,以后生长减缓;当茎叶枯萎时,根中生物量短期快速积累。与常规水分条件相比,干旱胁迫处理显著降低了黄芪苗高及茎叶生物量,但对抗氧化能力、根系生长及次生代谢物积累产生了不同的影响。轻度干旱胁迫下SOD、POD、CAT 3种抗氧化酶活性升高,丙二醛(MDA)含量和细胞膜透性降低,同时根长与根生物量增加、多糖与皂苷两种次生代谢物积累增多,黄芪药材的质量得到显著提高(P0.05);胁迫上升到中度、重度时,SOD酶活性逐渐降低,重度胁迫下低于对照,而POD及CAT酶活性、MDA含量、细胞膜透性均随胁迫增强而升高,相反,根长、根生物量、多糖与皂苷含量降低,导致黄芪药材的质量显著降低(P0.01)。综上表明,干旱胁迫下,SOD酶表现较为敏感,可能在清除活性氧中起主要作用;轻度水分胁迫能有效启动黄芪体内抗氧化酶系统和次生代谢,它们相互协作共同对抗胁迫对细胞产生的伤害,通过降低地上部分的生长,将营养物质优先运往根部,促进根产量及药材质量的提高。这一结论,可在黄芪多糖和皂苷次生代谢物定向培育的水分管理中加以利用。     

干旱胁迫对喀斯特地区野生茶树幼苗生理特性及根系生长的影响

以贵州喀斯特地区4种野生茶树(Sect Thea(L.)Dyer)无性系幼苗为材料,采用盆栽控水方法研究其对干旱胁迫的生理生长响应,初步评价其抗旱性,并采用田间模拟持续干旱试验进行验证。结果表明:(1)随着干旱胁迫程度的加强,4种野生茶树叶片的相对含水量逐渐降低;细胞质膜透性、丙二醛(MDA)含量和可溶性糖含量均呈上升趋势;游离脯氨酸含量和超氧化物歧化酶(SOD)活性先升后降;过氧化物酶(POD)活性除秃房茶随胁迫程度加强呈上升趋势外,其他种均呈先升后降趋势。(2)随着干旱胁迫程度的加强,4种茶树幼苗总生物量干重均逐渐下降,根冠比先升后降;根系总长除在秃房茶中呈先升后降趋势外,在其他3个种中均逐渐下降;根系总表面积、根系总体积在大理茶和茶中逐渐下降,而在大厂茶和秃房茶中先升后降;根系平均直径在秃房茶中逐渐降低,大厂茶中先升后降,茶中逐渐升高;根系活力在大理茶中逐渐下降,在其他3种中均先升后降;而比叶面积则在大厂茶中呈下降趋势,在其他3种中均先升后降。(3)基于生长生理指标的隶属函数抗旱性评价结果表明,MDA含量和细胞质膜透性与野生茶树抗旱性密切相关,4种野生茶树幼苗的抗旱顺序为秃房茶(Camellia gymnogyna Chang)大厂茶(Camellia tachangensis F.C.Zhang)茶(Camellia Sinensis(L.)-O.Kuntze)大理茶(Camellia taliensis(W.W.Smish)),田间持续干旱试验验证了基于隶属函数抗旱性评价方法的准确性和可靠性。     

Expression of an Arabidopsis vacuolar H+‐pyrophosphatase gene (AVP1) in cotton improves drought‐ and salt tolerance and increases fibre yield in the field conditions

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up‐regulation of the type I H⁺‐PPase from Arabidopsis may result in a higher proton electrochemical gradient, which facilitates enhanced sequestering of ions and sugars into the vacuole, reducing water potential and resulting in increased drought‐ and salt tolerance when compared to wild‐type plants. Furthermore, overexpression of AVP1 stimulates auxin transport in the root system and leads to larger root systems, which helps transgenic plants absorb water more efficiently under drought conditions. Using the same approach, AVP1‐expressing cotton plants were created and tested for their performance under high‐salt and reduced irrigation conditions. The AVP1‐expressing cotton plants showed more vigorous growth than wild‐type plants in the presence of 200 mm NaCl under hydroponic growth conditions. The soil‐grown AVP1‐expressing cotton plants also displayed significantly improved tolerance to both drought and salt stresses in greenhouse conditions. Furthermore, the fibre yield of AVP1‐expressing cotton plants is at least 20% higher than that of wild‐type plants under dry‐land conditions in the field. This research indicates that AVP1 has the potential to be used for improving crop’s drought‐ and salt tolerance in areas where water and salinity are limiting factors for agricultural productivity.     

Antioxidant Responses of Lentil to Cold and Drought Stress

The effects of cold and drought stress on antioxidant responses and growth parameters in shoots and roots of lentil (Lens culinaris M cv Sultan 1) seedlings were investigated. Ten-day-old hydroponically grown seedlings were subjected to drought and cold (4°C) stress for 5 days. The length and fresh weight of shoots decreased significantly under both stress conditions, contrary to the increase in these growth parameters for roots under the same conditions. The oxidative damage as generation of malondialdehyde and hydrogen peroxide, was markedly higher in shoots under cold. Both stress conditions caused a significant increase in malondialdehyde levels in root tissues. The increase in proline levels was more pronounced under cold stress in shoots and roots. The tested stress conditions had no significant effect on chlorophyll contents. Superoxide dismutase activity was differentially altered in shoot and root tissues under drought and cold stress. The catalase activity was higher in roots under drought stress. On the other hand, ascorbate peroxidase activity increased in root tissues under cold stress. The results indicate that improved tolerance to cold and drought stress in root and shoot tissues of lentil might be correlated to the increased capacity of antioxidative defense system.     

干旱条件下接种AM真菌对小马鞍羊蹄甲幼苗根系的影响

为了探讨岷江干旱河谷丛枝菌根真菌(AMF)对寄主植物幼苗根系的影响,通过接种购买的AMF摩西球囊霉菌(Funneliformis mosseae)到优势乡土灌木小马鞍羊蹄甲(Bauhinia faberi var.microphylla)幼苗,在重度、中度和轻度干旱条件下培养3个月,研究不同干旱条件下AMF对幼苗根系形态特征、结构特征、功能性状的影响。方差分析结果表明:(1)3种干旱胁迫条件下,接菌均显著增加了幼苗的根总长、根表面积、根分枝数、根尖数(P0.001),在中度胁迫和轻度胁迫下,接菌显著促进根鲜重、根体积的增加(P0.001),轻度胁迫条件下接菌幼苗的根鲜重、根总长、根表面积、根体积、根尖数最高并显著高于其它处理,但接菌与未接菌的根平均直径之间没有显著差异;(2)接菌幼苗根系趋向于叉状分支结构,在重度胁迫时,叉状分支趋势更显著(P0.001);(3)接菌幼苗的根比例都显著小于未接菌的,但幼苗比根长不存在显著差异。相关分析结果表明:菌根侵染率与根鲜重、根总长、根表面积、根体积、根分枝数、根尖数呈极显著正相关(P0.001),与拓扑指数、根比例呈极显著负相关(P0.001)。研究表明,在干旱条件下,AMF虽然没有提高生长初期的根系的吸收效率,但接种AMF显著影响幼苗根系形态特征和结构特征,更利于植物适应干旱环境,并且AMF对幼苗根系的促生作用随着干旱胁迫程度减轻而提高。     

交替灌溉对山黧豆叶片气体交换和土壤水分以及产量指标的影响

改变土壤根系的分布以汲取深层土壤水分的能力是植物避免干旱的主要策略。山黧豆是一种抗逆性强的豆类作物,该研究通过起垄条播控制性沟灌的方式,设置传统灌溉(FI)、交替灌溉(PRD,灌水量减少50%)和不灌溉(NI)3种处理模式,探索不同灌溉模式对播种后不同时期山黧豆土壤水分、根系分布、叶片气体交换、水分利用效率和籽粒产量的影响。结果表明：(1)在FI、PRD和NI处理下,山黧豆的根系分别有89.8%、86.9%和84.9%生长在0~20 cm的表层土壤中;干旱胁迫使PRD和NI处理下深层土壤中根系的比例提高至13.05%和15.07%。(2)在整个生育期内,土壤干旱显著降低了山黧豆叶片的净光合速率、蒸腾速率和气孔导度;在种植后60 d时,PRD和NI处理下叶片的瞬时水分利用效率分别较FI处理显著提高了21.4%和14.9%。(3)干旱胁迫显著降低了山黧豆植株高度、第一豆荚高、平均结荚数和豆粒数以及地上部和根系的干重,但显著增加了根冠比;PRD处理对豆荚长度、豆荚重和每荚豆粒重没有显著影响;PRD和NI处理下山黧豆平均籽粒产量分别比FI处理显著降低了53%和63%。研究发现,在干旱胁迫条件下,山黧豆能够通过提高深层土壤中根系的比例、更多吸收深层土壤水分、显著增加根冠比以及显著提高生殖生长期叶片的瞬时水分利用效率,减轻干旱胁迫对自身生长的影响。该研究结果可为山黧豆在旱区推广种植提供理论依据。     

拟南芥ABA敏感突变体asm1的分离与鉴定

以拟南芥(Arabidopsis thaliana)为研究材料,从T-DNA突变体库中筛选分离得到1株脱落酸(ABA)敏感突变体asm1(ABA sensitive mutant 1,asm1),在含有ABA的培养基中,与野生型相比,asm1突变体的根伸长明显受到抑制,且其种子萌发结果显示asm1对ABA同样表现出敏感特性。在生长发育方面,asm1突变体抽苔时间提前,植株矮化,并且荚果长度明显小于野生型。利用远红外成像系统分析发现,在干旱胁迫下asm1突变体叶面温度高于野生型;失水率分析显示突变体失水率降低以及水分散失减少。遗传学分析表明,asm1是单基因隐性突变且与一个T-DNA插入共分离;通过图位克隆成功获得候选基因ASM1。RT-PCR结果显示,在突变体中ASM1的表达受到抑制,并且能够调控多种ABA信号通路和胁迫应答基因的表达水平。研究结果表明,ASM1可能参与调控ABA信号转导并应答干旱胁迫。     

Paenibacillus lentimorbus Enhanced Abiotic Stress Tolerance Through Lateral Root Formation and Phytohormone Regulation

Under the stressed conditions plant growth-promoting rhizobacteria (PGPR) are able to stimulate plant growth through several mechanisms, including antioxidants alleviation, regulation of stress responsive genes and phytohormones etc. Present study is conducted to investigate the impact of Paenibacillus lentimorbus B-30488 inoculation on salinity and drought stress mitigation in Arabidopsis thaliana through modulation in defense enzymes, phyto-hormones and root system architecture associated gene expression profiling. In vitro experiments clearly demonstrated the role of B-30488 in stimulating the root length, branches, lateral root formation and biomass under salinity and drought stress. The inoculation of B-30488 modulated the phytohormones levels to protect the plants from salinity and drought stress. Similarly, defence enzymes were also activated under the stressed conditions, but B-30488 inoculation reduced the antioxidants content during salinity and drought stress as compared to their respective controls. Microscopy results showed decrease in lateral roots hair formation under both stresses and B-30488 inoculation not only mitigate but also enhanced the lateral root formation. Gene expression analysis through real time polymerase chain reaction (RT-PCR) showed modulated expression of several genes related to root development, stress and lateral root formation in B-30488 inoculated seedlings. Results based on the present study, B-30488 is also involved in alteration root architecture, its growth regulation via modulation in phytohormones and genes expression and overall significant improvement in plant growth under stress conditions.

     

An osmotin from the resurrection plant Tripogon loliiformis (TlOsm) confers tolerance to multiple abiotic stresses in transgenic rice

Osmotin is a key protein associated with abiotic and biotic stress response in plants. In this study, an osmotin from the resurrection plant Tripogon loliiformis (TlOsm) was characterized and functionally analyzed under abiotic stress conditions in T. loliiformis as well as in transgenic Nicotiana tabacum (tobacco) and Oryza sativa (rice) plants. Real‐time PCR analysis on mixed elicitor cDNA libraries from T. loliiformis showed that TlOsm was upregulated a 1000‐fold during the early stages of osmotic stresses (cold, drought, and salinity) in both shoots and roots but downregulated in shoots during heat stress. There was no change in TlOsm gene expression in roots of heat‐stressed plants and during plant development. The plasma membrane localization of TlOsm was showed in fluorescent‐tagged TlOsm tobacco plants using confocal laser scanning microscopic analysis. Transgenic rice plants expressing TlOsm were assessed for enhanced tolerance to salinity, drought and cold stresses. Constitutively expressed TlOsm in transgenic rice plants showed increased tolerance to cold, drought and salinity stress when compared with the wild‐type and vector control counterparts. This was evidenced by maintained growth, retained higher water content and membrane integrity, and improved survival rate of TlOsm‐expressing plants. The results thus indicate the involvement of TlOsm in plant response to multiple abiotic stresses, possibly through the signaling pathway, and highlight its potential applications for engineering crops with improved tolerance to cold, drought and salinity stress.     

Maize root responses to drought stress depend on root class and axial position

In this study we tested the hypotheses that root classes would exhibit distinctive anatomical and architectural responses to drought stress, and that those responses would vary along the root axes. The root systems of four maize (Zea mays L.) sweet corn genotypes designated SC1, SC2, SC3 and SC4 were phenotyped under well-watered and drought treatments in greenhouse mesocosms, permitting increasing stratification of moisture availability as the drought progressed. Anatomical and architectural responses to drought were evaluated for each root class. Lignin distribution was assessed by image processing of UV-illuminated root cross-sections acquired by laser ablation tomography. The two cultivars with less biomass reduction under drought, SC3 and SC4, substantially enhanced lateral root development along the apical segments of axial roots when plants were grown with drought stress. These segments grew into the deeper part of the mesocosm where more moisture was available. Apical segments of the axial and large lateral roots from drought-stressed plants were thicker and had greater theoretical axial water conductance than basal segments, especially in SC3 and SC4. Basal segments of crown roots of SC3 and SC4 showed increased lignification of the stele under drought. Root anatomical and architectural responses to drought are complex and vary among cultivars and root classes, and along root axes. Drought-induced proliferation of lateral roots on apical segments of axial roots would be expected to enhance deep water acquisition, while lignification of axial roots could help preserve axial water transport.

     

不同水分胁迫对绵毛水苏幼苗形态和生理特性的影响

以绵毛水苏幼苗为试材,以正常供水为对照,研究不同水分胁迫(淹水、渍水、中度干旱、重度干旱)对绵毛水苏形态和生理指标的影响。结果表明:(1)绵毛水苏植株在淹水处理3d后外围叶片坏死,根系死亡,但茎基部有不定根萌生;渍水处理植株地上部始终无明显变化,但部分根系根尖变褐色;干旱处理7d时叶片萎蔫,且重度干旱处理叶片萎蔫程度大于中度干旱。胁迫解除后,除淹水处理在第13天恢复生长外,其余处理均在第2天恢复正常生长。(2)绵毛水苏叶片含水量和根系活力在淹水处理下显著降低,在渍水处理下无明显变化;在干旱胁迫下,叶片含水量迅速下降,而根系活力升高。(3)在各水分胁迫条件下,绵毛水苏植株叶、根的相对电导率和MDA含量均较对照显著升高,且根部受损程度均重于叶片,其中淹水胁迫受损最严重。(4)淹水处理叶片可溶性蛋白含量下降,可溶性糖、游离脯氨酸持续积累;渍水和干旱处理叶和根的可溶性糖、可溶性蛋白和游离脯氨酸均升高。研究认为,绵毛水苏具有较强的渗透调节能力,在渍水和干旱胁迫解除后迅速缓解膜质过氧化伤害,恢复正常生长;绵毛水苏虽不能在长时间淹水条件下生长,但可在渍水条件下正常生长,且能忍受干旱胁迫,可应用于滨水消落带等水分变化较大的区域。