RNAi‐mediated Ultra‐low gossypol cottonseed trait: performance of transgenic lines under field conditions

Cottonseed remains a low‐value by‐product of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. Ultra‐low gossypol cottonseed (ULGCS) lines were developed using RNAi knockdown of δ‐cadinene synthase gene(s) in Gossypium hirsutum. The purpose of the current study was to assess the stability and specificity of the ULGCS trait and evaluate the agronomic performance of the transgenic lines. Trials conducted over a period of 3 years show that the ULGCS trait was stable under field conditions and the foliage/floral organs of transgenic lines contained wild‐type levels of gossypol and related terpenoids. Although it was a relatively small‐scale study, we did not observe any negative effects on either the yield or quality of the fibre and seed in the transgenic lines compared with the nontransgenic parental plants. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. As expected, the major difference between the ULGCS and wild‐type cottonseeds was in terms of their gossypol levels. With the exception of oil content, the composition of ULGCS was similar to that of nontransgenic cottonseeds. Interestingly, the ULGCS had significantly higher (4%–8%) oil content compared with the seeds from the nontransgenic parent. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi‐based product has the potential to be commercially viable. Thus, it may be possible to enhance and expand the nutritional utility of the annual cottonseed output to fulfil the ever‐increasing needs of humanity.     

RNAi suppression of CYP82D P450 hydroxylase,an enzyme involved in gossypol biosynthesis,enhances resistance to Fusarium wilt in cotton

Cotton plants produce two classes of terpenoid defence compounds against pathogens and other pests. Both classes are derived from a common sesquiterpenoid precursor, δ-cadinen-2-one, which enters either the gossypol pathway or the lacinilene pathway. Blocking the gossypol pathway by RNAi suppression of the early pathway biosynthetic enzyme CYP82D hydroxylase resulted in enhanced resistance to the Fusarium wilt pathogen. Analyses of root terpenoids revealed no overall increases in the products of the gossypol pathway in the roots infected by the wilt pathogen. However, the lacinilene pathway was elicited by the pathogen and the lacinilene levels were 19-fold higher in the RNAi plants than in wild-type plants. In the pathogen inoculated RNAi 73R plants, the concentrations of DHC and HMC were 231 μg and 886 μg/g dry roots, respectively, which may have contributed to the inhibition of fungal invasion. In comparison, the concentrations of DHC and HMC in the pathogen inoculated control wild-type 73W plants were only 0.7 μg and 58 μg/g dry roots, respectively. Fungitoxicity testing showed that DHC at 100 μg/ml inhibited growth of the Fusarium wilt pathogen by >93%. Treatment with the phytohormone jasmonic acid failed to elicit production of lacinilene pathway terpenoids in roots of either RNAi plants or their wild-type sibling lines, but increased production of gossypol pathway terpenoids with concentrations in RNAi plants 80%–97% less than those in wild-type plants. This indicates that induction of the lacinilene pathway is not directly mediated by jasmonic acid signalling and requires other signalling to activate the pathway. These results illustrate possible mechanisms of wilt disease resistance in cotton and provide a new approach to increase host resistance by manipulating these two major cotton chemical defence pathways.     

Terpenoid aldehyde formation and lysigenous gland storage sites in cotton: variant with mature glands but suppressed levels of terpenoid aldehydes

A new cotton variant with reduced levels of terpenoid aldehydes (sesquiterpenoids and sesterterpenoids (heliocides)) was isolated from the progeny of hemizygous cotton (Gossypium hirsutum cv. Coker 312) transformed with antisense (+)-delta-cadinene synthase cDNA. Southern analysis of leaf DNA digested with HindIII, Pst or KpnI restriction endonucleases did not detect any antisense cdn1-C1 DNA in the genome of the variant. The gossypol content in the seed of the variant was markedly lower than in the seed of T1 antisense plants. Eighty-nine percent of the variant seed had a 71.1% reduction in gossypol and the foliage of the variant plants showed a 70% reduction in gossypol and a 31% reduction in heliocides. Compared to non-transformed plants there was no reduction in the number of lysigenous glands in the seed of the variant. The cotton variant shows uncoupling of terpenoid aldehyde synthesis and gland formation. The cotton variant may have resulted from somaclonal variation occurring in the callus tissue during the transformation-regeneration process.     

Vermicompost treatment differentially affects seed germination,seedling growth and physiological status of vegetable crop species

Vermicompost preparations are increasingly used in agricultural practice. There is a possibility, that crop plants are sensitive to negative effect of vermicompost at early stages of development. The aim of the present study was to test the effects of vermicompost on seed germination and seedling growth of different vegetable crop species. Vermicompost substitution inhibited seed germination and seedling growth with almost linear decrease of growth with increasing concentration of vermicopost in the substrate. However, both leaf chlorophyll content and photochemical activity of photosynthesis increased in all crop species with the exception of pea seedlings. Vermicompost extract as a watering solution showed positive effect on growth of bean and pea seedlings. Germination response of vermicompost extract-imbibed seeds was clearly crop species-dependent. Hypocotyl growth was stimulated by low and moderate vermicompost extract concentrations. Radicle growth was more sensitive to negative effect of vermicompost extract. It is reported that both solid vermicompost and vermicompost extract contain number of active substances of both phenolic and humic nature, each with own dose- and genotype-dependent effect of seed germination and early stages of seedling development. Findings of this study suggests that vermicompost must be used cautiously for practical purposes of plant propagation.     

The underground phase: a special challenge in studies of terrestrial orchid populations

Orchid seeds are minute and the first seedling stages of terrestrial species are underground, so there is little knowledge about the biology of the early life history and the size of seed/seedling populations relative to the number of plants that eventually emerge above ground. A recently developed field sowing technique makes it possible to explore a variety of topics such as the length of time that seeds spend as part of the soil seed bank, the phenology of seed germination, substrate requirements for germination, dependence of germination on fungal availability, substrate requirements and growth rate of seedlings, seedling phenology and development, and seedling-fungus specificity patterns in the soil. Several of these applications are relevant for conservation issues. Studies of Goodyera pubescens, Corallorhiza odontorhiza and Liparis lilifolia show considerable variation in germination strategies, patterns of seedling development and establishment, substrate requirements, and levels of specificity with the fungi.     

四种植物种子萌发及苗期抗旱性差异的研究

对四种豆科植物种子萌发及苗期抗旱性作了比较研究,结果表示：种子萌发期抗旱笥强的植物其苗期抗旱性亦强。干旱引起组织脱水,植株生长受到抑制,幼苗出现衩始萎蔫时间与细胞膜受害时间一致。四种植物显示萎蔫时土壤含水量明显不同,红豆草为４．８０％、羊柴２．２％、花棒１．７４％、柠条１．５１％．据种子相对发芽率、膜透性变化等生理指标综合评定四种植物种子萌发及苗期抗旱能力次序为：柠条＞花棒＞羊柴＞红豆草。     

干旱胁迫下华北驼绒藜种子大小及苞片对萌发和幼苗生长的影响

干旱是影响荒漠区植物种子萌发和幼苗生长的关键因素.以多年生强旱生半灌木华北驼绒藜为对象,研究了不同干旱程度(0、100、200、300和400g·L-1PEG6000)下,种子大小及苞片有无对种子萌发及幼苗生长的影响.结果表明:干旱胁迫显著抑制了种子萌发和幼苗地上部的生长.100和200 g ? L-1 PEG6000...     

OsLOX2, a rice type I lipoxygenase,confers opposite effects on seed germination and longevity

Rice production and seed storage are confronted with grain deterioration and loss of seed viability. Some members of the lipoxygenase (LOX) family function in degradation of storage lipids during the seed germination, but little is known about their influence on seed longevity during storage. We characterized the role of rice OsLOX2 gene in seed germination and longevity via over-expression and knock-down approaches. Abundant expression of OsLOX2 was detected in panicles, roots, and stems, but not in leaves. Moreover, OsLOX2 was highly induced during germination. OsLOX2 protein, located in the cytoplasm, showed a wide range of temperature adaptation (20–50 °C) and a substrate preference to linoleic acid. Lines over-expressing OsLOX2 showed accelerated seed germination under normal condition and lower seed viability after accelerated aging. RNA interference (RNAi) of OsLOX2 caused delayed germination and enhanced seed longevity. RNAi lines with strongly repressed OsLOX2 activity completely lost the capability of germination after accelerated aging. More lipid hydroperoxide were found in OE15 than the control, but less in RNAi lines than in the WT Nipponbare. Therefore, OsLOX2 acts in opposite directions during seed germination and longevity during storage. Appropriate repression of the OsLOX2 gene may delay the aging process during the storage without compromising germination under normal conditions.     

杉木种子与幼苗不同发育期的耐旱性研究

以广东杉木(Cunninghamia lanceolata)第二代改良种子园种子为试材,利用不同浓度聚乙二醇(PEG-6000)溶液(5%、10%、20%和40%)模拟不同程度(轻度、中度、较强、重度)干旱胁迫,并对不同干旱条件下的种子萌发、幼苗子叶期和真叶期不同发育阶段的形态变化进行测定分析。结果发现,杉木种子萌发初期对水分尤为敏感,耐旱性不强,尤其是随干旱程度的加剧,种子起始萌发时间不断后延且发芽率显著降低。子叶后期的杉木幼苗较子叶前期耐旱,真叶期的杉木幼苗对轻度和中度干旱胁迫具有较强的耐受性。     

EARLY RESPONSIVE TO DEHYDRATION 15, a negative regulator of abscisic acid responses in Arabidopsis

EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15) is rapidly induced in response to various abiotic and biotic stress stimuli in Arabidopsis (Arabidopsis thaliana). Modulation of ERD15 levels by overexpression or RNAi silencing altered the responsiveness of the transgenic plants to the phytohormone abscisic acid (ABA). Overexpression of ERD15 reduced the ABA sensitivity of Arabidopsis manifested in decreased drought tolerance and in impaired ability of the plants to increase their freezing tolerance in response to this hormone. In contrast, RNAi silencing of ERD15 resulted in plants that were hypersensitive to ABA and showed improved tolerance to both drought and freezing, as well as impaired seed germination in the presence of ABA. The modulation of ERD15 levels not only affected abiotic stress tolerance but also disease resistance: ERD15 overexpression plants showed improved resistance to the bacterial necrotroph Erwinia carotovora subsp. carotovora accompanied with enhanced induction of marker genes for systemic acquired resistance. We propose that ERD15 is a novel mediator of stress-related ABA signaling in Arabidopsis.     

南亚热带森林24种乔木的种子萌发和幼苗生长

以膨胀珍珠岩为基质,在光和暗的条件下,对２４种南亚热带森林乔木的种子萌发和幼苗生长进行了研究。种子的形态和重量与种的演替阶段有关,种子的重量也与不同的种有关。不同种的萌发率差异较大,种子较大的种,萌发率较高。肉质果实的种子,开始萌发的时间较长。光和暗条件对萌发率和萌发速度无明显影响。幼苗高度和种子重量呈正相关,这一相关在光条件下比暗条件下更明显。暗条件明显地引起群落演替早期的树种的茎徒长,限制根的生长,而对演替后期种无显著的影响。幼苗地上部分和根系生物量的分配与种的演替阶段有关,也与不同的科有关。幼苗地上部分和根系生物量的分配也受光暗条件的影响,而不受种子重量的影响。根和叶的生物量分配和种在其群落演替阶段有关。幼苗的生物量与种子重量有显著的正相关。幼苗的相对生长率相差较大,在没有营养供应的生长基质中,以演替中间阶段的种的相对生长率较大。幼苗从种子的物质利用效率与种子重量呈负相关,而与种的演替阶段无明显的相关。     

Ectopic expression of ABSCISIC ACID 2/GLUCOSE INSENSITIVE 1 in Arabidopsis promotes seed dormancy and stress tolerance

Abscisic acid (ABA) is an important phytohormone that plays a critical role in seed development, dormancy, and stress tolerance. 9-cis-Epoxycarotenoid dioxygenase is the key enzyme controlling ABA biosynthesis and stress tolerance. In this study, we investigated the effect of ectopic expression of another ABA biosynthesis gene, ABA2 (or GLUCOSE INSENSITIVE 1 [GIN1]) encoding a short-chain dehydrogenase/reductase in Arabidopsis (Arabidopsis thaliana). We show that ABA2-overexpressing transgenic plants with elevated ABA levels exhibited seed germination delay and more tolerance to salinity than wild type when grown on agar plates and/or in soil. However, the germination delay was abolished in transgenic plants showing ABA levels over 2-fold higher than that of wild type grown on 250 mm NaCl. The data suggest that there are distinct mechanisms underlying ABA-mediated inhibition of seed germination under diverse stress. The ABA-deficient mutant aba2, with a shorter primary root, can be restored to normal root growth by exogenous application of ABA, whereas transgenic plants overexpressing ABA2 showed normal root growth. The data reflect that the basal levels of ABA are essential for maintaining normal primary root elongation. Furthermore, analysis of ABA2 promoter activity with ABA2::beta-glucuronidase transgenic plants revealed that the promoter activity was enhanced by multiple prolonged stresses, such as drought, salinity, cold, and flooding, but not by short-term stress treatments. Coincidently, prolonged drought stress treatment led to the up-regulation of ABA biosynthetic and sugar-related genes. Thus, the data support ABA2 as a late expression gene that might have a fine-tuning function in mediating ABA biosynthesis through primary metabolic changes in response to stress.     

Do seeds sense each other? Testing for density‐dependent germination in desert perennial plants

The timing of seedling emergence may strongly affect fitness in competitive environments. Therefore, selection should favour mechanisms that allow sensing neighbours prior to emergence. We tested whether or not germination is affected by density and identity of neighbouring seeds or seedlings of desert perennial plants. Based on theory, we predicted that germination fractions are independent of neighbouring seeds, that germination is accelerated in dense interspecific neighbourhoods, and neighbour effects are caused by seedlings, not by seeds. We examined germination fraction and timing of four naturally coexisting sandy desert perennial species in low versus high seed densities in both intra- and interspecific neighbourhoods, and with and without removal of newly emerged seedlings. Neighbours accelerated germination independent of density and this pattern was apparently caused by the presence of early emerging seedlings. Germination fractions were lower in high seed densities even when neighbours did not germinate, indicating that seeds were able to sense each other prior to emergence. Early germination may be adaptive because fast emerging seedlings may gain a competitive edge over slow emerging ones. However, since seeds that did not germinate died, delayed germination may only be advantageous for mother plants when sib competition is intense. Another key finding was a competitive hierarchy with late successional species germinating faster and inhibiting germination of pioneer species. This indicates that successional processes may be directed as early as during germination.     

植物的防御性萜类挥发信号分子

植物生长发育过程中会合成大量萜类化合物,其中多数具有挥发性。这些挥发性萜类成分除参与保护光合器官免受光合作用产生的热量灼伤之外,还作为信号分子参与了大量的化学生态过程,例如对昆虫和其他动物的吸引以利于花粉和种子的传播。另一方面,自上世纪80年代以来,逐渐有研究工作表明,挥发性萜类化合物也可以作为信号介导植物对植食性昆虫的防御反应,以及在植物．害虫．天敌的三级营养关系中发挥作用。同时,研究工作还显示萜类化合物可以作为信号在同株植物的不同部位之间及邻近植物之间进行有效传递以促进对虫害的防御,并证明了这些萜类分子在细胞和分子水平上的调控作用。这些工作为利用化学生态学手段对虫害进行有效控制提供了新的策略。文章仅就近年来对植物萜类挥发信号分子在合成、识别、传输及其作用等方面的研究进展做简要回顾。     

萜类合成酶定向进化的新思路

萜类化合物是天然产物中种类最多且主要存在于植物和微生物体内的一类化合物。随着越来越多具有应用价值的萜类化合物被挖掘,其应用前景引起了人们的关注,但由于含量低、提取成本高等缺点,因此制约了萜类化合物的广泛应用。合成生物学的兴起,为异源合成具有应用价值的萜类化合物提供了新思路,使构建定向、高效的微生物细胞工厂成为现实。萜类合成酶常作为萜类化合物异源合成代谢调控的靶酶,但天然的萜类合成酶存在催化效率低、底物专一性差、立体/区域选择性差、稳定性差等问题,严重影响萜类化合物的产量。萜类合成酶的定向进化可以有效地解决上述问题,为实现微生物细胞工厂异源、高效合成萜类化合物奠定基础。本文综述了近年来酶的定向进化技术的最新进展及应用,并提出了萜类合成酶定向进化的策略。