Knockdown of NtMed8, a Med8-like gene, causes abnormal development of vegetative and floral organs in tobacco (Nicotiana tabacum L.)

Med8, a subunit of mediator complex, has proved to possess crucial functions in many organisms from yeast to human. In plant, the med8 mutant of Arabidopsis thaliana displayed delayed anthesis and increased number of leaves during the vegetative period. However, the roles of Med8 in other flowering plants are still unknown. To investigate the function of Med8 ortholog in tobacco (Nicotiana tabacum L.; named as NtMed8), we created transgenic tobacco plants with repressed NtMed8 expression mediated by RNA interference (RNAi). Compared with the wild type, the NtMed8-RNAi plants exhibited: more leaves with smaller but thicker blades; larger cells and vascular bundles with lower stomata density in leaves; swelled chloroplasts with thicker and lumen-enlarged thylakoids; weaker root system with fewer lateral roots; larger flowers and floral organs; flowering earlier under long day, but later under short day conditions; and male sterile with larger but less germinable pollens. In addition, quantitative RT-PCR indicated that NtMed8 is expressed in both vegetative and floral tissues. Subcellular localization analysis by transient expression of fusion protein in Nicotiana benthamiana leaves showed that NtMed8 was located in both plasma membrane and nucleus. These results suggest that NtMed8 plays important roles in both vegetative and reproductive development, and the function of Med8 appears to be, at least partially, conserved in flowering plants.     

The maize d2003, a novel allele of VP8, is required for maize internode elongation

The d2003 is a natural dwarf mutant from maize inbred line K36 and has less than one-third of K36 plant height with severely shortened internodes. In this study, we reported the cloning of d2003 gene using positional cloning. The results showed that there was a single-base insertion in the coding region of Viviparous8 (VP8) in d2003 mutant, which resulted in a premature stop codon. Further genetic allelism tests confirmed that d2003 mutation is a novel allele of VP8. VP8 is mainly expressed in the stem apex, young leaves, and developing vascular tissues, and its expression levels in nodes are significantly higher than that in internodes at 12-leaf stage. Subcellular localization demonstrated that the VP8 protein is localized to the endoplasmic reticulum and the N-terminal 26 amino acids (aa) of VP8 protein are essential to its localization in ER. Further transgenic experiments showed that lack of the 26 aa leads to loss of VP8 function in Arabidopsis amp1 phenotype rescue. These results strongly suggested that the N-terminal 26 aa is critical for VP8 protein localization, and the correct protein localization of VP8 in ER is necessary for its function.     

AtCML8, a calmodulin-like protein, differentially activating CaM-dependent enzymes in Arabidopsis thaliana

Plants express many calmodulins (CaMs) and calmodulin-like (CML) proteins that sense and transduce different Ca²⁺ signals. Previously, we reported divergent soybean (Glycine max) CaM isoforms (GmCaM4/5) with differential abilities to activate CaM-dependent enzymes. To elucidate biological functions of divergent CaM proteins, we isolated a cDNA encoding a CML protein, AtCML8, from Arabidopsis. AtCML8 shows highest identity with GmCaM4 at the protein sequence level. Expression of AtCML8 was high in roots, leaves, and flowers but low in stems. In addition, the expression of AtCML8 was induced by exposure to salicylic acid or NaCl. AtCML8 showed typical characteristics of CaM such as Ca²⁺-dependent electrophoretic mobility shift and Ca²⁺ binding ability. In immunoblot analyses, AtCML8 was recognized only by antiserum against GmCaM4 but not by GmCaM1 antibodies. Interestingly, AtCML8 was able to activate phosphodiesterase (PDE) but did not activate NAD kinase. These results suggest that AtCML8 acts as a CML protein in Arabidopsis with characteristics similar to soybean divergent GmCaM4 at the biochemical levels.     

Leaf development and photosynthetic properties of three tropical tree species with delayed greening

Leaf developmental patterns were characterized for three tropical tree species with delayed greening. Changes in the pigment contents, photosynthetic capacity, stomata development, photosystem 2 efficiency, rate of energy dissipation, and the activity of partial protective enzymes were followed in developing leaves in an attempt to elucidate the relative importance of various photoprotective mechanisms during leaf ontogeny. Big leaves of Anthocephalus chinensis, a fast-growing light demanding species, expanded following an exponential pattern, while relatively small leaves of two shade-tolerant species Litsea pierrei and Litsea dilleniifolia followed a sigmoidal pattern. The juvenile leaves of A. chinensis and L. pierrei contained anthocyanin located below the upper epidermis, while L. dilleniifolia did not contain anthocyanin. Leaves of A. chinensis required about 12 d for full leaf expansion (FLE) and photosynthetic development was delayed 4 d, while L. pierrei and L. dilleniifolia required 18 or 25 d for FLE and photosynthetic development was delayed 10 or 15 d, respectively. During the leaf development the increase in maximum net photosynthetic rate was significantly related to changes in stomatal conductance and the leaf maturation period was positively related to the steady-state leaf dry mass per area for the three studied species. Dark respiration rate of leaves at developing stages was greater, and pre-dawn initial photochemical efficiency was lower than that of mature leaves. Young leaves displayed greater energy dissipation than mature leaves, but nevertheless, the diurnal photoinhibition of young L. dilleniifolia leaves was higher than that of mature leaves. The young red leaves of A. chinensis and L. pierrei with high anthocyanin contents and similar diurnal photoinhibition contained more protective enzymes (superoxide dismutase, ascorbate peroxidase) than mature leaves. Consequently, red leaves may have higher antioxidant ability.     

巨桉扩展蛋白EgrEXPA8和EgrEXPA10基因的克隆和表达特性分析

为探讨扩展蛋白在桉树生长发育中的作用,以在桉树初生生长到次生生长转换转录组测序中筛选出的差异表达基因EgrEXPA8和EgrEXPA10为基础,从巨桉(Eucalyptusgrandis)中克隆了2个扩展蛋白基因EgrEXPA8和EgrEXPA10,分别编码249和244个氨基酸,属于亲水蛋白,但Egr EXPA8稳定性高于Egr EXPA10。q RT-PCR分析表明,Egr EXPA8和Egr EXPA10基因均在幼叶和茎尖组织中表达量较高,在木质部和韧皮部表达量较低;且在茎顶端初生生长阶段表达量较高,而在下部次生生长节间表达量较低,可能其主要参与巨桉的初生生长或者负调控次生生长;另外在盐胁迫、茉莉酸甲酯处理下其均被抑制表达;而在水杨酸、缺硼、缺磷处理下均上调表达。这说明EgrEXPA8和EgrEXPA10在巨桉响应逆境胁迫时起到重要作用,且呈现出相似的调控方式。     

Molecular cloning and characterization of novel isoforms of potato ADP-glucose pyrophosphorylase

ADP-glucose pyrophosphorylase (AGPase) is one of the major enzymes involved in starch biosynthesis in higher plants. We report here the molecular cloning of two cDNAs encoding so far uncharacterized isoforms (AGP S2 and AGP S3) of the potato enzyme. Sequence analysis shows that the two polypeptides are more homologous to previously identified large subunit polypeptides from potato and other plant species than to small subunit isoforms. This observation suggests that AGP S2 and AGP S3 represent novel large subunit polypeptides. agpS2 is expressed in several tissues of the potato plant, including leaves and tubers. Expression was stronger in sink leaves than in source leaves, indicating developmental regulation. In leaves, agpS2 expression was induced 2- to 3-fold by exogenous sucrose; therefore, agpS2 represents a new sucrose-responsive gene of starch metabolism. Expression of agpS3 was restricted to tubers: no agpS3 expression could be seen in leaves of different developmental stages, or when leaves were incubated in sucrose. Therefore, agpS3 represents the only AGPase gene so far characterized from potato, which is not expressed in leaves. Conversely, all four AGPase isoforms known from potato are expressed in tubers.     

3种外源诱导剂预处理对甜瓜抗病特征及其Fom 2和CHT基因表达的影响

该研究选用水杨酸(SA)、茉莉酸甲酯(MeJA)、Ca~(2+)、无菌水(对照)作为外源预处理诱导剂,以抗、感枯萎病甜瓜品种为材料,分别于诱导预处理2d后接种甜瓜枯萎菌,并于接种5、7、9d时观察发病情况,进行病情调查;在接种后1、3、5、7、9d取甜瓜叶片,分析抗病甜瓜(MR-1)和感病甜瓜(M1-15)叶片中甜瓜抗枯萎病基因(Fom-2)、几丁质酶基因(CHT)的表达变化,以探寻提高防治甜瓜枯萎病菌侵染的技术途径。结果显示:(1)外源MeJA和SA预处理接种后2品种的病情指数显著低于对照,但Ca~(2+)处理后的病情指数与对照无显著差异。(2)经外源诱导预处理接种后,MR-1和M1-15品种叶片的Fom-2和CHT基因均出现差异表达,但Ca~(2+)诱导其上调表达的效果微弱。(3)经SA、MeJA诱导预处理接种后,2品种叶片的Fom-2和CHT基因表达总体均显著高于对照;Fom-2基因的表达抗病甜瓜MR-1分别在接种后5d、7d时达到峰值,而感病甜瓜M1-15则均在接种9d时达到峰值;CHT基因的表达抗病甜瓜MR-1则均在接种后7d时达到峰值,而感病甜瓜M1-15分别在接种后7d、9d时达到峰值。(4)Ca~(2+)处理对抗、感甜瓜叶片的Fom-2和CHT基因的表达均无显著影响。(5)相关分析表明,经SA、MeJA诱导预处理接种后,甜瓜枯萎病病情指数与Fom-2和CHT基因表达量有显著的相关性;而Ca~(2+)处理效果不显著。研究表明:SA、MeJA通过诱导Fom-2、CHT基因上调表达,进而使甜瓜的抗病性提高,而Ca~(2+)处理对两基因表达和甜瓜抗病性均无显著影响。     

Transient expression, purification and characterization of bioactive human fibroblast growth factor 8b in tobacco plants

cDNA of human fibroblast growth factor 8 isoform b (FGF8b) was cloned for the first time into a plant expression vector with or without endoplasmic reticulum retention signal (KDEL) and was transiently expressed as His tagged fusion protein in Nicotiana tabacum leaves through Agrobacterium mediated gene transfer by vacuum infiltration method. Expression of FGF8b was confirmed by ELISA and Western blot using anti-FGF8b antibody and the expression level was measured as 4.1% of total soluble protein of tobacco leaves. The expressed recombinant protein was purified by Ni-NTA affinity chromatography and its molecular weight was determined by MALDI-TOF-MS. Schiff’s test, Concanavalin A (Con A) immunoblot and enzymatic deglycosylation indicated that the high molecular mass was due to glycosylation of the FGF8b expressed in plant cells. Measurement of its biological activity in NIH3T3 cells by thymidine incorporation and MTT assay showed induction of cell proliferation. These results indicate that biologically active recombinant FGF8b could be expressed in tobacco plants. Surya Kumar Potula and Sonal Roy Kathuria contributed equally to this work.     

The Rice OsRad21-4, an Orthologue of Yeast Rec8 Protein,is Required for Efficient Meiosis

In yeast, Rad21/Scc1 and its meiotic variant Rec8 are key players in the establishment and subsequent dissolution of sister chromatid cohesion for mitosis and meiosis, respectively, which are essential for chromosome segregation. Unlike yeast, our identification revealed that the rice genome has 4 RAD21-like genes that share lower than 21% identity at polypeptide levels, and each is present as a single copy in this genome. Here we describe our analysis of the function of OsRAD21-4 by RNAi. Western blot analyses indicated that the protein was most abundant in young flowers and less in leaves and buds but absent in roots. In flowers, the expression was further defined to premeiotic pollen mother cells (PMCs) and meiotic PMCs of anthers. Meiotic chromosome behaviors were monitored from male meiocytes of OsRAD21-4-deficient lines mediated by RNAi. The male meiocytes showed multiple aberrant events at meiotic prophase I, including over-condensation of chromosomes, precocious segregation of homologues and chromosome fragmentation. Fluorescence in situ hybridization experiments revealed that the deficient lines were defective in homologous pairing and cohesion at sister chromatid arms. These defects resulted in unequal chromosome segregation and aberrant spore generation. These observations suggest that OsRad21-4 is essential for efficient meiosis.     

Tannin Dynamics of Propagules and Leaves of Kandelia candel and Bruguiera gymnorrhiza in the Jiulong River Estuary, Fujian, China

Changes in the total phenolics, condensed tannins (CT), protein-precipitable phenolics content and protein precipitation capacity were determined on a series of mangrove leaves from two true viviparous mangrove species (Kandelia candel and Bruguiera gymnorrhiza) at various stages of development and decomposition in the Jiulong River Estuary, Fujian, China. Similar measurements were also done for the propagules at different developmental stages. The results showed that the total phenolics, extractable condensed tannins, total condensed tannins, protein-precipitable phenolics content and protein precipitation capacity in young leaves were higher than those in mature and senescent leaves. Tannin dynamics during leaf decomposition varied with species, and the rapid loss of phenolics observed during decomposition can be ascribed to leaching and degradation. Protein-bound CT and fibre-bound CT tended to increase with leaf decomposition, with CT binding more strongly to protein than to fibre. Protein-bound CT was higher than fibre-bound CT with the exception of mature leaves. Total phenolics, extractable CT and protein-precipitable phenolics contents in flower tissues were relatively lower than those in hypocotyls at different developmental stages. Protein precipitation capacity fluctuated with the development of propagules. Increases in nitrogen in decaying litter, and declines in contents of total phenolics and total condensed tannins of detritus support the general conclusion that decomposing mangrove detritus can be a more palatable heterotrophic substrate than living leaves.     

Arabidopsis casein kinase 1-like 8 enhances NaCl tolerance,early flowering,and the expression of flowering-related genes

Members of the casein kinase 1 (CK1) family are evolutionarily conserved eukaryotic protein kinases involved in various cellular, physiological, and developmental processes in yeast. However, the biological roles of CK1 members in plants are poorly understood. Here, we report that an Arabidopsis CK1 member named casein kinase 1-like 8 (CKL8) was ubiquitously expressed in all plant organs, mainly in the stems of seedlings according to quantitative real-time PCR. Western blotting showed a remarkable expression of the AtCKL8 gene in transgenic plants induced by high salinity. A histochemical assay of AtCKL8 promoter::GUS expression revealed that the AtCKL8 promoter is very active in both seedlings and adult plants subjected to the salinity stress, while no GUS activity was detectable in all the transgenic plants grown under normal conditions. In a subcellular distribution analysis, the AtCKL8-GFP fusion protein was localized mainly in the cell membrane. AtCKL8-overexpressing transgenic plants showed an insensitivity to high salinity and an early flowering phenotype. Overall, these findings suggest that AtCKL8 plays a positive role in NaCl signaling and improves salt stress tolerance in transgenic Arabidopsis.     

转GhB301基因烟草的防御酶活性及抗病相关基因表达分析

为了明确棉花ERF-B3亚族转录因子基因GhB301在烟草异位表达后(抗枯萎病中)的功能,该研究以过表达GhB301基因烟草和野生型烟草为材料,采用枯萎病菌孢子悬浮液接菌方法,分析病原菌侵染前后防御酶活性变化以及防卫相关基因的表达变化与抗病性的关系。结果显示:(1)棉花枯萎病菌处理15d后,2个转基因株系烟草叶片黄化程度与野生型相比较轻。(2)棉花枯萎病菌处理后,过表达GhB301转基因烟草和野生型烟草叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性较未接菌对照显著提高,并且酶活峰值出现均早于野生型材料;转基因材料叶片的POD、PAL、PPO活性均在处理3d后达到峰值,而野生型材料叶片的POD、PAL活性在处理5d后才达到峰值。(3)接种棉花枯萎病菌后活性氧相关基因、乙烯(ET)/茉莉酸(JA)途径相关基因、病程相关基因的表达量在转基因株系OE1和OE2中均受到明显影响。研究推测,GhB301在烟草中的异位表达激活了防卫相关基因的表达,提高了防御酶的活性,从而增强了烟草对枯萎病菌的抗性。     

拟南芥莲座叶片发育过程中P1基因的表达特性

该实验对CDF1类似蛋白基因(P1)在拟南芥叶片发育不同阶段的定量PCR结果显示,P1基因在拟南芥叶片发育的所有时期均可表达,但在茎生叶和衰老叶中的表达水平明显高于成熟叶和幼叶。GUS报告基因表达的组织化学染色结果显示,P1启动子在拟南芥叶片中有较高的驱动活性;在营养生长阶段的幼苗和植株(4~5周)的所有叶片中均能检测到GUS表达,但在植株转入生殖生长阶段后(6周及以后),GUS表达主要集中在逐渐衰老的叶中,并随着叶片衰老程度加剧GUS染色程度也越深,这一结果与GUS荧光定量检测结果一致。通过分析P1基因启动子上可能存在的顺式调控元件,发现茉莉酸甲酯、热压、干旱和水杨酸等均能够引起叶片衰老调控元件的响应,证实P1的表达受到这些因素的调控。研究表明,P1在拟南芥莲座叶片中很可能参与了对上游衰老信号的响应,该研究结果为进一步探究P1在叶片衰老过程中的分子功能验证奠定了基础。