The<Emphasis Type="Italic"> Arabidopsis thaliana rlp</Emphasis> mutations revert the ectopic leaf blade formation conferred by activation tagging of the<Emphasis Type="Italic"> LEP</Emphasis> gene

Activation tagging of the gene LEAFY PETIOLE ( LEP) with a T-DNA construct induces ectopic leaf blade formation in Arabidopsis, which results in a leafy petiole phenotype. In addition, the number of rosette leaves produced prior to the onset of bolting is reduced, and the rate of leaf initiation is retarded by the activation tagged LEP gene. The ectopic leaf blade results from an invasion of the petiole region by the wild-type leaf blade. In order to isolate mutants that are specifically disturbed in the outgrowth of the leaf blade, second site mutagenesis was performed using ethane methanesulphonate (EMS) on a transgenic line that harbours the activation-tagged LEP gene and exhibits the leafy petiole phenotype. A collection of revertant for leafy petiole ( rlp) lines was isolated that form petiolated rosette leaves in the presence of the activated LEP gene, and could be classified into three groups. The class III rlp lines also display altered leaf development in a wild-type (non-transgenic) background, and are probably mutated in genes that affect shoot or leaf development. The rlp lines of classes I and II, which represent the majority of revertants, do not affect leaf blade outgrowth in a wild-type (non-transgenic) background. This indicates that LEP regulates a subset of the genes involved in the process of leaf blade outgrowth, and that genetic and/or functional redundancy in this process compensates for the loss of RLP function during the formation of the wild-type leaf blade. More detailed genetic and morphological analyses were performed on a selection of the rlp lines. Of these, the dominant rlp lines display complete reversion of (1) the leafy petiole phenotype, (2) the reduction in the number of rosette leaves and (3) the slower leaf initiation rate caused by the activation-tagged LEP gene. Therefore, these lines are potentially mutated in genes for interacting partners of LEP or in downstream regulatory genes. In contrast, the recessive rlp lines exhibit a specific reversion of the leafy petiole phenotype. Thus, these lines are most probably mutated in genes specific for the outgrowth of the leaf blade. Further functional analysis of the rlp mutations will contribute to the dissection of the complex pathways underlying leaf blade outgrowth.Communicated by G. Jürgens     

T-DNA插入水稻群体中卷叶突变体R1-A2的遗传分析

在根癌农杆菌介导的T-DNA(携带有除草剂Basta抗性基因bar和Ds因子)转化中花11水稻群体中,获得了一个叶片发生明显内卷的突变体R1-A。经过连续三代的分离鉴定,获得突变体的纯合株(R1-A2),并与中花11号进行杂交,在调查的36个F_1植株中,全部表现为卷叶,并对Basta除草剂都表现为抗性。在852个F_2单株中,卷叶为645株,正常叶207株,卷叶和正常叶的比例为3:1,其中,卷叶株均对Basta表现抗性,正常叶株均对Basta表现敏感,表明卷叶性状和Basta抗性存在着共分离关系。用扩增Ds因子的引物,对F_2中45个卷叶抗性株进行PCR鉴定,都获得预期长度的Ds因子片段,进一步表明在这些卷叶的植株中都有T-DNA的插入;而30个正常叶敏感株都不能检测到Ds的特征片段。在以卷叶突变(R1-A2)为回交亲本的F_1B_1植株中,全部植株表现卷叶;在以中花11号为回交亲本的F_1B_1植株中,卷叶和正常叶植株的分离比为1:1。上述结果表明该卷叶突变是个显性突变,受一个基因所控制,且该基因的突变与T-DNA的插入有关。     

T—DNA插入水稻群体中卷叶突变体R1—A2的遗传分析

在根癌农杆菌介导的T-DNA(携带有除草剂Basta抗性基因bar和Ds因子)转化中花11水稻群体中,获得了一个叶片发生明显内卷的突变体Rl-A。经过连续三代的分离鉴定,获得突变体的纯合株(Rl-A2),并与中花11号进行杂交,在调查的36个F1植株中,全部表现为卷叶,并对Basta除草剂都表现为抗性。在852个F2单株中,卷叶为645株,正常叶207株,卷叶和正常叶的比例为3：1,其中,卷叶株均对Basta表现抗性,正常叶株均对Basta表现敏感,表明卷叶性状和Basta抗性存在着共分离关系。用扩增DS因子的引物,对F2中45个卷叶抗性株进行PCR鉴定,都获得预期长度的Ds因子片段,进一步表明在这些卷叶的植株中都有T-DNA的插入;而30个正常叶敏感株都不能检测到DS的特征片段。在以卷叶突变(Rl-A2)为回交亲本的F1B1植株中,全部植株表现卷叶;在以中花11号为回交亲本的F1B1植株中,卷叶和正常叶植株的分离比为1：1。上述结果表明该卷叶突变是个显性突变,受一个基因所控制,且该基因的突变与T-DNA的插入有关。     

Anatomical Injury Induced by the Eriophyid Mite Aceria anthocoptes on the Leaves of Cirsium arvense

Anatomical injury of the leaves of the invasive species, Cirsium arvense (L.) Scop., caused by the eriophyid mite Aceria anthocoptes (Nal.), which is the only eriophyid mite that has been recorded on C. arvense worldwide, is described. The injury induced by the mite feeding on the leaves of C. arvense results in visible russeting and bronzing of the leaves. Other conspicuous deformations are folding and distortion of the leaf blade and curling of leaf edge, as well as gradual drying of leaves. The anatomical injury of the mature leaves of field-collected plants was limited to the epidermis of the lower leaf surface. However, on young leaves of experimentally infested plants, rust mite injuries extend to epidermal cells on both leaf surfaces and to those of deeper mesophyll layers. On these leaves, lesions on the lower leaf surface even affected the phloem of the vascular bundles. Leaf damage induced by A. anthocoptes is discussed with regard to the mite’s potential as a biological control agent of C. arvense.     

<Emphasis Type="Italic">NARROW LEAF 7</Emphasis> controls leaf shape mediated by auxin in rice

Elucidation of the genetic basis of the control of leaf shape could be of use in the manipulation of crop traits, leading to more stable and increased crop production. To improve our understanding of the process controlling leaf shape, we identified a mutant gene in rice that causes a significant decrease in the width of the leaf blade, termed narrow leaf 7 (nal7). This spontaneous mutation of nal7 occurred during the process of developing advanced backcrossed progeny derived from crosses of rice varieties with wild type leaf phenotype. While the mutation resulted in reduced leaf width, no significant morphological changes at the cellular level in leaves were observed, except in bulliform cells. The NAL7 locus encodes a flavin-containing monooxygenase, which displays sequence homology with YUCCA. Inspection of a structural model of NAL7 suggests that the mutation results in an inactive enzyme. The IAA content in the nal7 mutant was altered compared with that of wild type. The nal7 mutant overexpressing NAL7 cDNA exhibited overgrowth and abnormal morphology of the root, which was likely to be due to auxin overproduction. These results indicate that NAL7 is involved in auxin biosynthesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under accession numbers AB354301 and AB354302.     

A putative AGO protein,OsAGO17, positively regulates grain size and grain weight through OsmiR397b in rice

Argonaute (AGO) proteins and small RNAs (sRNAs) are core components of the RNA‐induced silencing complex (RISC). It has been reported that miRNAs regulate plant height and grain size in rice, but which AGO is involved in grain size regulation remains unclear. Here, we report that enhanced expression of OsAGO17, a putative AGO protein, could improve grain size and weight and promote stem development in rice. Cytological evidence showed that these effects are mainly caused by alteration of cell elongation. Expression analyses showed that OsAGO17 was highly expressed in young panicles and nodes, which was consistent with the expression pattern of OsmiR397b. SRNA sequencing, stem‐loop RT‐PCR and sRNA blotting showed that the expression of OsmiR397b was reduced in ago17 and enhanced in the OsAGO17 OE lines. Four OsmiR397b target laccase (LAC) genes showed complementary expression patterns with OsAGO17 and OsmiR397b. Combined with the results of immunoprecipitation (IP) analysis, we suggested that OsAGO17 formed an RISC with OsmiR397b and affected rice development by suppression of LAC expression. In conclusion, OsAGO17 might be a critical protein in the sRNA pathway and positively regulates grain size and weight in rice.     

Factors affecting the rate of T-DNA transfer from Agrobacterium tumefaciens to Nicotiana glauca plant cells

Different factors involved in the early steps of the T-DNA transfer process were studied by using a -glucuronidase gene (gusA) as a reporter in Nicotiana glauca leaf disc transformation experiments. The levels of transient expression of the gusA gene in leaf discs infected with several strains or vir mutants correlated well with their virulence phenotype, except for virC mutants. The rate of T-DNA transfer was shown to be stimulated in the case of non-oncogenic strains by the co-transfer of small amounts of oncogenic genes. It was found that the location of the T-DNA in the Agrobacterium genome affected the T-DNA transfer rate especially in virC mutants. The virC mutants transferred the gusA-containing T-DNA located on a binary vector more efficiently than the oncogenic T-DNA of the Ti plasmid. Although wild-type strains induced high levels of gusA expression early after infection, the gusA expression appeared to be lost late after infection in the infected leaf discs. In contrast, in leaf discs infected by virC mutants the level of gusA expression increased steadily in time. A model explaining these results is presented.     

Constitutively wilted 1, a member of the rice YUCCA gene family, is required for maintaining water homeostasis and an appropriate root to shoot ratio

Increasing its root to shoot ratio is a plant strategy for restoring water homeostasis in response to the long-term imposition of mild water stress. In addition to its important role in diverse fundamental processes, indole-3-acetic acid (IAA) is involved in root growth and development. Recent extensive characterizations of the YUCCA gene family in Arabidopsis and rice have elucidated that member’s function in a tryptophan-dependent IAA biosynthetic pathway. Through forward- and reverse-genetics screening, we have isolated Tos17 and T-DNA insertional rice mutants in a CONSTITUTIVELY WILTED1 (COW1) gene, which encodes a new member of the YUCCA protein family. Homozygous plants with either a Tos17 or T-DNA-inserted allele of OsCOW1 exhibit phenotypes of rolled leaves, reduced leaf widths, and lower root to shoot ratios. These phenotypes are evident in seedlings as early as 7–10 d after germination, and remain until maturity. When oscow1 seedlings are grown under low-intensity light and high relative humidity, the rolled-leaf phenotype is greatly alleviated. For comparison, in such conditions, the transpiration rate for WT leaves decreases approx. 5- to 10-fold, implying that this mutant trait results from wilting rather than being a morphogenic defect. Furthermore, a lower turgor potential and transpiration rate in their mature leaves indicates that oscow1 plants are water-deficient, due to insufficient water uptake that possibly stems from that diminished root to shoot ratio. Thus, our observations suggest that OsCOW1-mediated IAA biosynthesis plays an important role in maintaining root to shoot ratios and, in turn, affects water homeostasis in rice.     

Pinnately ramified ensiform leaves in the genus Marathrum, (Podostemaceae-Podostemoideae)

The paper deals with the complex leaves of three species of the aquatic genus Marathrum (M. minutiflorum, M. rubrum and M. schiedeanum). It is shown that they represent pinnately ramified ensiform leaves. The dissection of the leaves contributes to surface enlargement and thus to improvement of their hydrophylls function. The ensiform structure is confirmed by the transverse position of the sheathing lower leaf zone and the subsequent intercalary elongational growth proceeding in the median plane towards the abaxial side of the leaf (upper leaf zone). The petiole continues into the rachis-like central axis of the blade from which pinnate-like segments diverge at the adaxial and abaxial edge of the ensiform blade. The segments give rise to tufts of clavate enations that end in long filaments. The filigree pinnately segmented ensiform leaves of Marathrum are interpreted as a further development of the fan-shaped and irregularly lobate ensiform leaves occurring in Mourera and Apinagia. Pinnately ramified ensiform leaves obviously evolved in convergence to the common type of pinnate leaves found in angiosperms.     

RAPD analysis of blast resistant somaclones from upland rice cultivar IAC 47 for genetic divergence

Seventeen somaclones of upland rice cultivar IAC 47 showing different plant types, and either resistance or susceptibility to leaf blast, were utilized for random amplified polymorphic DNA (RAPD) analysis. Somaclones exhibited differences in reaction to isolates of Pyricularia grisea. Two somaclones (SC02 and SC04) were resistant to all three field isolates of somaclones, while the cultivar IAC 47 was susceptible. The inheritance study of two distinct plant types, one with erect bright green leaves and the other with droopy yellow green leaves, showed that a single possibly different, dominant gene governs each plant type. Of 32 random decamer primers utilized, OPA02 and OPD02 detected polymorphisms between somaclones showing erect bright green leaves and droopy yellow green leaves. Reliable grouping exhibiting 80% similarity was achieved with 17 primers. Leaf blast resistance to race IC-2 of P. grisea was associated with the plant type of erect bright green leaves.     

Monitoring leaf photosynthesis with canopy spectral reflectance in rice

Non-destructive and rapid method for assessment of leaf photosynthetic characteristics is needed to support photosynthesis modelling and growth monitoring in crop plants. We determined the quantitative relationships between leaf photosynthetic characteristics and canopy spectral reflectance under different water supply and nitrogen application rates. The responses of reflectance at red radiation (wavelength 680 nm) to different water contents and nitrogen rates were parallel to those of leaf net photosynthetic rate (P _N). The relationships of reflectance at 680 nm and ratio index of R(810,680) (near infrared/red, NIR/R) to P _N of different leaf positions and leaf layers in rice indicated that the top two full leaves were the best leaf positions for quantitative monitoring of leaf P _N with remote sensing technique, and the ratio index R(810,680) was the best ratio index for evaluating leaf photosynthetic characteristics in rice. Testing of the models with independent data sets indicated that R(810,680) could well estimate P _N of top two leaves and canopy leaf photosynthetic potential in rice, with the root mean square error of 0.25, 0.16, and 4.38, respectively. Hence R(810,680) can be used to monitor leaf photosynthetic characteristics at different growth stages of rice under diverse growing conditions.     

The relationship between freezing tolerance and thermotropic leaf movement in five Rhododendron species

Summary Leaf movement kinetics in five species of Rhododendron were studied in response to leaf temperature, leaf freezing point, and leaf water deficit. There was a gradient in the degree of leaf curling among species in the following order from the greatest curling to the least curling: Rhododendron catawbiense, R. maximum, R. minus, R. macrophyllum, R. ponticum. Those species found to be tolerant of winter conditions had the most intense leaf movements (both curling and angle) while those species with minimal cold tolerance had limited or no leaf movements. Leaf curling occurred at leaf temperatures above the tissue freezing points in all species. Athough leaf angle was influenced by leaf turgor, general tissue desiccation was not the ultimate cause for thermotropic leaf curling in any species tested. Those species with the greatest leaf curling and angle movements had the highest osmotic potential, the lowest water deficit at the turgor loss point, and the lowest symplastic water fraction. These data suggest that there is a trade off in Rhododendron leaf physiology between cold tolerance (due to leaf movements) and water stress tolerance (due to turgor maintenance mechanisms).     

ARGONAUTE 2 increases rice susceptibility to rice black-streaked dwarf virus infection by epigenetically regulating HEXOKINASE 1 expression

ARGONAUTE (AGO) proteins play crucial roles in plant defence against virus invasion. To date, the role of OsAGO2 in rice antiviral defence remains largely unknown. In this study, we determined that the expression of OsAGO2 in rice was induced upon rice black-streaked dwarf virus (RBSDV) infection. Using transgenic rice plants overexpressing OsAGO2 and Osago2 mutants generated through transposon-insertion or CRISPR/Cas9 technology, we found that overexpression of OsAGO2 enhanced rice susceptibility to RBSDV infection. Osago2 mutant lines exhibited strong resistance to RBSDV infection through the elicitation of an early defence response, including reprogramming defence gene expression and production of reactive oxygen species (ROS). Compared to Nipponbare control, the expression level of OsHXK1 (HEXOKINASE 1) increased significantly, and the methylation levels of its promoter decreased in the Osago2 mutant on RBSDV infection. The expression profile of OsHXK1 was the opposite to that of OsAGO2 during RBSDV infection. Overexpression of OsHXK1 in rice also induced ROS production and enhanced rice resistance to RBSDV infection. These results indicate that OsHXK1 controls ROS accumulation and is regulated by OsAGO2 through epigenetic regulation. It is noteworthy that the Osago2 mutant plants are also resistant to southern rice black-streaked dwarf virus infection, another member of the genus Fijivirus. Based on the results presented in this paper, we conclude that OsAGO2 modulates rice susceptibility to fijivirus infection by suppressing OsHXK1 expression, leading to the onset of ROS-mediated resistance. This discovery may benefit future rice breeding programmes for virus resistance.     

A novel alkaline α-galactosidase gene is involved in rice leaf senescence

We previously isolated and identified numerous senescence-associated genes (SAGs) in rice leaves. Here we characterized the structure and function of an SAG-Osh69 encoding alkaline α-galactosidase that belongs to a novel family of glycosyl hydrolases. Osh69 is a single-copy gene composed of 13 exons located on rice chromosome 8. The expression level of Osh69 is not only up-regulated during natural leaf senescence but also induced rapidly by darkness, hormones (methyl jasmonic acid, salicylic acid), and stresses (H₂O₂ and wounding). The recombinant Osh69 protein over-expressed in Escherichia coli has displayed optimal α-galactosidase activity at pH 8.0. The enzyme showed good hydrolytic activities towards α-1,6-galactosyl oligosaccharides and galactolipid digalactosyl diacylglycerol. Immunoelectron microscopic analysis demonstrates that Osh69 is specifically localized in the chloroplasts of senescing leaves. These findings strongly suggest an important role for Osh69 in the degradation of chloroplast galactolipids during leaf senescence. The nucleotide sequence data reported will appear in the GenBank Nucleotide Sequence Database under the accession number AF251068.     

小立碗藓PpAGO7基因启动子分离及其启动活性分析

AGO7蛋白在多种植物中被发现并预测在叶片生长发育过程中起作用,但是在高等植物中最古老且唯一没有维管束的苔藓植物中却未有报道。该文通过BLAST比对水稻OsAGO7基因编码的氨基酸序列得到小立碗藓AGO7蛋白编码基因PpAGO7,扩增PpAGO7基因起始密码子上游的启动子序列,利用在线软件PlantCARE和PLACE分析该启动子的结构特征;并构建启动子分析载体pPpAGO7-GUS,转化拟南芥,通过转基因拟南芥GUS染色结果分析推测PpAGO7启动子的启动特性。结果显示:(1)PpAGO7基因启动子序列中含有大量的光反应有关的顺式作用元件以及数个分生组织相关和防御与胁迫相关作用元件。(2)T2代转基因拟南芥的GUS染色结果表明,PpAGO7基因启动子会启动GUS在拟南芥的不同部位和不同生长时期表达,而且在根尖、叶片顶端、雄蕊的花药、雌蕊的柱头和种子等部位的染色较其他部位更深。(3)生长在光照强度1 000lx和4 000lx下转基因拟南芥的GUS酶活性比光照强度7 000lx和10 000lx下的低。研究表明所克隆的PpAGO7基因启动子具有组成性启动活性,且在生长旺盛的部位启动活性较强,此外其启动活性还受到光照因素的影响,为进一步研究小立碗藓的PpAGO7基因功能提供了重要依据。     

一份水稻叶片反卷突变体的遗传分析及电镜显微观察

叶片是植物进行光合作用的重要器官。叶片适度卷曲能够提高水稻(Oryza sativa)生长中后期群体基部的光能利用率, 因而有利于水稻产量的提高。该研究首先在水稻T-DNA插入突变体库中发现一份叶片反卷的突变体。遗传分析表明, 该性状受到1对隐性核基因控制。扫描电镜观察结果显示, 突变体成熟叶片上下表皮的气孔发生了畸变; 且叶片上表皮气孔数目增多, 而下表皮气孔数目与野生型基本相同。叶片横切面电镜观察结果表明, 与野生型相比, 突变体叶片的泡状细胞数目和面积在早期(二叶期)就开始增加, 在成熟期更加明显, 这可能是导致叶片反卷的主要原因。     

巨桉不同状态叶片浸提液对萝卜幼苗形态和抗性生理特性的影响

以珍珠岩作为基质,选择4年生巨桉(Eucalyptus grandis)嫩叶(T1)、老叶(T2)、表层凋落叶(T3)、腐解凋落叶(T4)4种状态的叶片,每种状态叶片设置3个浸提液浓度水平[分别称取风干叶片30g、15g和7.5g加入900mL蒸馏水进行浸提,以蒸馏水为对照(CK)],采用水培法研究了不同状态叶片浸提液对萝卜(Raphanus sativus)幼苗形态生长和抗性生理特性的影响。结果显示:(1)巨桉不同状态叶片浸提液显著抑制了萝卜幼苗的根长,其中嫩叶的抑制作用最强,腐解凋落叶抑制作用最弱。(2)各状态叶片浸提液处理后萝卜幼苗中过氧化氢酶(CAT)和过氧化物酶(POD)的活性均呈现升高趋势,嫩叶各浓度处理以及其他状态叶片的高浓度处理下超氧化物歧化酶(SOD)活性升高,而其余浓度处理的SOD活性降低。(3)各状态叶片浸提液处理萝卜幼苗的丙二醛(MDA)含量在低浓度处理时低于CK,其余处理下则高于CK。(4)嫩叶各浓度处理萝卜幼苗的可溶性糖(SS)含量显著高于CK,且随着老叶和表层凋落叶浸提液浓度的升高,幼苗SS含量先升后降,腐解凋落叶各浓度处理下则呈渐增的趋势;而可溶性蛋白(SP)含量则随浸提液浓度的增加而升高,且T2和T3两种状态叶片的各浓度处理与CK差异显著。研究表明,巨桉不同状态叶片浸提液对萝卜幼苗生长和抗性生理产生了强烈的抑制作用,其中以嫩叶最强,老叶和表层凋落叶次之,腐解凋落叶最弱。     

Leaf Longevity,Construction, and Maintenance Costs of Three Mangrove Species Under Field Conditions

This study assessed the effect of leaf age on construction cost (CC) in the mangrove species Avicennia germinans, Laguncularia racemosa, and Rhizophora mangle growing in their natural habitat. Leaf osmolality values were species-specific, the highest in A. germinans (1 693 mmol kg^–1) and the lowest in L. racemosa (1 270 mmol kg^–1). In the three species, contents of chlorophyll (a+b) (Chl_a+b) and nitrogen (N) per unit of leaf area were maximal in adult leaves and tended to decline with age. Leaf mass to leaf area ratio (LMA) and ash content increased during leaf ageing. Similarly, as leaves aged, a significant increase in leaf construction cost per leaf area (CC_a) was observed, while per leaf mass (CC_m) it remained almost constant, suggesting a sustained production of leaf compounds as leaves became older. CC was positively correlated with LMA and heat of combustion (Hc) per leaf area, suggesting differences among species in the quantity and composition of expensive compounds. Leaf half lifetime (t_0.5) showed contrasting values in the three mangrove species (60, 111, and 160 d in L. racemosa, R. mangle, and A. germinans, respectively). Overall, L. racemosa was the species with less expensive leaves to construct while leaves of A. germinans and R. mangle had the highest CC_m and CC_a, respectively. Leaf longevity was positively correlated with the ratio between CC and maximum photosynthetic rate (P _max), clearly showing the existence of a balance between leaf costs and benefits.