Ultrastructure of Mesophyll Cells and Pigment Content in Senescing Leaves of Maize and Barley

Leaf senescence is a genetically regulated stage in the plant life cycle leading to death. Ultrastructural analysis of a particular region of the leaf and even of a particular mesophyll cell can give a clear picture of the time development of the process. In this study we found relations between changes in mesophyll cell ultrastructure and pigment concentration in every region of the leaf during leaf senescence in maize and barley. Our observations demonstrated that each mesophyll cell undergoes a similar senescence sequence of events: a) chromatin condensation, b) degradation of thylakoid membranes and an increase in the number of plastoglobules, c) damage to internal mitochondrial membrane and chloroplast destruction. Degradation of chloroplast structure is not fully correlated with changes in photosynthetic pigment content; chlorophyll and carotenoid content remained at a rather high level in the final stage of chloroplast destruction. We also compared the dynamics of leaf senescence between maize and barley. We showed that changes to the mesophyll cells do not occur at the same time in different parts of the leaf. The senescence damage begins at the base and moves to the top of the leaf. The dynamics of mesophyll cell senescence is different in leaves of both analyzed plant species; in the initial stages, the process was faster in barley whereas in the later stages the process occurred more quickly in maize. At the final stage, the oldest barley mesophyll cells were more damaged than maize cells of the same age.     

Natural developmental variations in leaf and plant senescence in Arabidopsis thaliana

Leaf senescence is a developmentally regulated process that contributes to nutrient redistribution during reproductive growth and finally leads to tissue death. Manipulating leaf senescence through breeding or genetic engineering may help to improve important agronomic traits, such as crop yield and the storage life of harvested organs. Here, we studied natural variations in the regulation of plant senescence among 16 Arabidopsis thaliana accessions. Chlorophyll content and the proportion of yellow leaves were used as indicator parameters to determine leaf and plant senescence respectively. Our study indicated significant genotype effects on the onset and development of senescence. We selected three late- and five early-senescence accessions for further physiological studies. The relationship between leaf and plant senescence was accession-dependent. There was a significant correlation between plant senescence and the total number of leaves, siliques and plant bolting age. We monitored expression of two senescence marker genes, SAG12 and WRKY53 , to evaluate progression of senescence. Our data revealed that chlorophyll content does not fully reflect leaf age, because even fully green leaves had already commenced senescence at the molecular level. Integrating senescence parameters, such as the proportion of senescent leaves, at the whole plant level provided a better indication of the molecular status of the plant than single leaf senescence parameters.     

Natural variation in the regulation of leaf senescence and relation to N and root traits in wheat

Objectives

To identify parameters that can be used for the analysis of natural variation in leaf senescence of wheat; and to understand the association between the onset and progression of leaf senescence with N uptake and root traits.

Methods

Chlorophyll content and the proportion of yellow leaves were used as senescence indicators and their relation with other morphological and physiological traits were measured in contrasting early senescing (ES) and late senescing (LS) wheat lines.

Results

There were significant genotype effects on the onset and progress of senescence. The ES lines in which leaf senescence commenced early had significantly lower root biomass and N uptake than LS lines. The strong negative association between the extent of leaf senescence with root biomass and N uptake indicated that the poor root growth induced N limitation caused the early senescence of ES lines.

Conclusions

The leaf senescence development in ES lines was precocious and constitutive as the trait expressed even under optimal growth conditions suggesting they could be useful in understanding the genetic regulation of senescence under different abiotic stress situations. Accelerated leaf senescence in wheat could be a mechanism to compensate for limitations in the root system that tend to restrict nutrient uptake.     

低氮诱导玉米幼苗叶片衰老过程中碳氮平衡的动态变化

叶片适时衰老对保证玉米产量有重要意义。本试验以玉米自交系PH6WC和PH4CV为研究对象,通过水培方法,设置低氮(0.04 mmol·L^-1,LN)和正常(4 mmol·L^-1,CK)氮素水平两种处理,在培养2、4、6和8 d后,对其幼苗第2和第3叶片表型、光合特性、叶片中氮素和糖分含量及碳氮比进行分析,旨在探究低氮胁迫下玉米幼苗叶片衰老过程中碳氮平衡的动态变化。结果表明: 与CK相比,LN造成两玉米自交系幼苗第2和第3叶片的面积、生物量、相对叶绿素含量、净光合速率、可溶性糖和淀粉含量均下降,而其氮物质生产能力均先后增加,但第2叶片的变化时间均早于第3叶片;在两叶片的各性状上,均为LN下PH6WC的变化幅度大于PH4CV,且仅幼苗叶片中的碳氮比在LN下显著提高;PH6WC的叶片衰老更快,PH4CV有更强的碳氮平衡能力,其叶片衰老相对滞后。综上,低氮会诱导玉米幼苗叶片衰老,高碳氮比具有促进叶片衰老的调控作用,低氮胁迫下幼苗叶片的碳氮平衡能力在两个玉米基因型间存在较大差异。     

Natural variation in the regulation of leaf senescence and relation to other traits in Arabidopsis

Leaf senescence results in the recycling of nutrients, thereby providing resources required for growth and reproduction. In this study, the effect of day-length on leaf senescence in eight different Arabidopsis thaliana ecotypes was determined and the relationship between senescence and other morphological and life history traits was analysed. A significant variation in the start and extent of leaf senescence depending on the genetic background and the response to day-length was found. Whereas senescence of early flowering ecotypes was accelerated by long days, no effect of day-length on senescence could be found in late flowering Kas-1 plants. Senescence in the different ecotypes was associated with other traits, such as floral transition, the total number of fruits, the total number of leaves and the maximum chlorophyll content. Plants that bolted early also senesced early, produced fewer leaves, accumulated less chlorophyll, but produced more fruits. The present results indicate that senescence may be a key component in the trade-off between investment in photosynthetic capacity and reproduction. The relationship between senescence and other traits was maintained independent of whether differences in senescence were caused by genetic (ecotype) or environmental (day-length) variation, suggesting that genetic and environmental factors affect these traits through common regulatory pathways.     

Changes in chloroplast peroxidase activities in relation to chlorophyll loss in barley leaf segments

Total peroxidase activity increased during senescence of excised barley ( Hordeum vulgare L. cv. Kashimamugi) leaves. Kinetin treatment furter increased total peroxidase activity but repressed chlorophyll degradation in excised barley leaves. When isoperoxidases were extracted from barley leaf segments. 4 cationic and 4 anionic isozymes were found in polyacrylamide gel electrophorests during leaf senescence. The chloroplasts contained only two cationic isoperoxidase activities. One (designated C4) was repressed by kinetin. and the other (C3) was increased by kinetin. Glucosamine, which also repressed the degradation of chlorophyll, completely repressed C4 activity but did not affect C3 activity. The induction with senescence, and the repression with kinetin and glucosamine, suggest chat chloroplast isoperoxidase C4 may function as a chlorophyll-degrading enzyme during barley leaf senescence.     

Lax midrib1-O, a systemic, heterochronic mutant of maize

Lxm1-O, a dominant EMS (ethyl methanesulfonate) induced mutation in maize (Zea mays, Poaceae), was originally reported to affect the blade/sheath boundary over the midrib region of the leaf. Here we present a more extensive analysis of the Lxm phenotype in nine different inbred lines. Lxm leaves are longer and narrower, and can initiate ectopic leaves. Additionally, Lxm1-O affects all plant organs observed. Compared to wild-type siblings, Lxm plants have fewer nodes, basal displacement of reproductive structures, and advance more quickly to the reproductive phase. We address questions as to whether Lxm1-O abbreviates a specific developmental phase, using hair, wax, and ear node data. We found that each phase was affected, although to varying degrees, depending on the inbred line. We interpret Lxm1-O to be a heterochronic mutation, causing the developmental acceleration of each phase of the shoot. Lxm1-O is novel, since other systemic heterochronic maize mutants prolong the juvenile phase, thereby extending shoot development. We discuss the importance of heterochronic mutations in the context of morphological evolution.     

干旱对夏玉米苗期叶片权衡生长的影响

叶片是植物对干旱响应最敏感的器官之一,叶片性状变化及其权衡关系能够反映植物对资源的利用策略以及对干旱的适应对策。基于2014年6个初始土壤水分梯度的夏玉米持续干旱模拟试验研究表明,随着干旱的发展,夏玉米各叶片性状均会受到影响,但不同干旱程度的影响不一致。基于水分胁迫系数及干旱持续时间提出了干旱程度的定量表达,随着干旱的发生发展,干旱程度在0—1之间变化。当干旱程度小于0.21时,夏玉米叶片性状不会受到显著影响;0.21—0.76时,叶片性状大小受到影响,但变化趋势不会发生改变;0.76—0.91时,新叶形成补偿不了老叶脱落,有效叶片数、叶干重、绿叶面积和叶含水量等性状提前出现下降趋势;大于0.91时,叶片生长几乎停滞。夏玉米叶片性状在干旱条件下的适应性生长本质上体现了其在快速生长与维持生存之间的权衡,但不同干旱程度下,夏玉米叶片性状生长的权衡策略不同:未发生干旱时,夏玉米倾向于维持较高的代谢活性,一旦干旱程度大于0,夏玉米就会降低叶片代谢活性;当干旱程度小于0.48时,夏玉米倾向于通过迅速增加叶面积来吸收较多的能量,以获得较大的生长速率,为生殖器官的生长及产量形成储备能量;当干旱程度大于0.48时,夏玉米会减小单叶面积以减少水分散失,倾向于资源贮存以提高其生存能力。     

Are isocitrate lyase and phosphoenolpyruvate carboxykinase involved in gluconeogenesis during senescence of barley leaves and cucumber cotyledons?

The aim of this study was to investigate whether gluconeogenesis catalysed by phosphoenolpyruvate carboxykinase (PEPCK) occurs during leaf senescence. This was addressed by determining changes in the abundance and intercellular location of enzymes necessary for gluconeogenesis during the senescence of barley leaves and cucumber cotyledons. PEPCK was never present in barley leaves, despite the presence of large amounts of isocitrate lyase (ICL), a key enzyme of the glyoxylate cycle, and of its product, glyoxylate. Although PEPCK was present in non-senescent cucumber cotyledons, its abundance declined during senescence. Throughout senescence, PEPCK was only present in the trichomes and vasculature, whereas ICL was located in mesophyll cells. Pyruvate,Pi dikinase (PPDK) which, in concert with NAD(P)-malic enzyme, is also capable of catalysing gluconeogenesis, was present in non-senescent barley leaves and cucumber cotyledons, but in both plants its abundance decreased greatly during senescence. The abundance of ICL was greatly reduced in senescing detached barley leaves by either illumination or by co-incubation with sucrose, and greatly increased in darkened attached barley leaves. These results argue against the large-scale occurrence of gluconeogenesis during senescence catalysed either by PEPCK or PPDK. In cucumber cotyledons, PEPCK may play a role in metabolic processes linked to the export of amino acids, a role in which phosphoenolpyruvate carboxylase may also be involved. The amount of ICL was increased by starvation and during senescence may function in the conversion of lipids to organic acids, which are then utilised in the mobilisation of amino acids from leaf protein.     

Aspects of programmed cell death during early senescence of barley leaves: possible role of nitric oxide

Summary. Leaf senescence is a highly coordinated process which involves programmed cell death (PCD). Early stages of leaf senescence occurring during normal leaf ontogenesis, but not triggered by stress factors, are less well known. In this study, we correlated condensation of chromatin and nuclear DNA (nDNA) fragmentation, two main features of PCD during early senescence in barley leaves, with the appearance of nitric oxide (NO) within leaf tissue. With the help of the alkaline version of the comet assay, together with measurements of nDNA fluorescence intensity, we performed a detailed analysis of the degree of nDNA fragmentation. We localised NO in vivo and in situ within the leaf and photometrically measured its concentration with the NO-specific fluorochrome 4-amino-5-methylamino-2′,7′-difluorofluorescein. We found that both nDNA fragmentation and chromatin condensation occurred quite early during barley leaf senescence and always in the same order: first nDNA fragmentation, in leaves of 6-day-old seedlings, and later chromatin condensation, in the apical part of leaves from 10-day-old seedlings. PCD did not start simultaneously even in neighbouring cells and probably did not proceed at the same rate. NO was localised in vivo and in situ within the cytoplasm, mainly in mitochondria, in leaves at the same stage as those in which chromatin condensation was observed. Localisation of NO in vascular tissue and in a large number of mesophyll cells during the senescence process might imply its transport to other parts of the leaf and its involvement in signalling between cells. The fact that the highest concentration of NO was found in the cytoplasm of mesophyll cells in the earliest stage of senescence and lower concentrations were found during later stages might suggest that NO plays an inductive role in PCD. Correspondence: A. Mostowska, Department of Plant Anatomy and Cytology, Institute of Experimental Biology of Plants, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland.     

Photosynthesis and leaf-nitrogen dynamics during leaf senescence of tropical maize cultivars in hydroponics in relation to N efficiency in the field

The selection process of nitrogen (N)-efficient cultivars during plant breeding could be simplified by a specification of secondary plant traits that are decisive for N efficiency. It was shown that leaf senescence under N deprivation of sixteen tropical maize cultivars in a short-term nutrient solution experiment was related to leaf senescence and grain yield under N deficiency (N efficiency) in field experiments. In this study we investigated if a quantification of leaf- and plant-N flows by ¹⁵N labelling can improve the evaluation of genotypic differences in leaf senescence in short-term experiments. Cultivars differed in leaf-N content prior to senescence; however, this appeared to have no significant impact on the development of leaf senescence. N import into senescing leaves was not related to total plant N uptake, but seems to have been regulated by leaf-inherent factors. Leaf N remaining in the leaf seems to have comprised inefficiently remobilized leaf N, at least during early senescence stages. Photosynthetic rate and chlorophyll contents at early senescence stages depended on additional factors to leaf-N content. Nevertheless, all parameters used to characterize leaf senescence were related to leaf senescence at anthesis in field experiments. However, only photosynthetic rate during late leaf senescence reflected cultivar differences in leaf senescence during reproductive growth and N efficiency in field experiments.     

Starch-branching enzyme IIa is required for proper diurnal cycling of starch in leaves of maize

Starch-branching enzyme (SBE), a glucosyl transferase, is required for the highly regular pattern of α-1,6 bonds in the amylopectin component of starch. In the absence of SBEIIa, as shown previously in the sbe2a mutant of maize (Zea mays), leaf starch has drastically reduced branching and the leaves exhibit a severe senescence-like phenotype. Detailed characterization of the maize sbe2a mutant revealed that SBEIIa is the primary active branching enzyme in the leaf and that in its absence plant growth is affected. Both seedling and mature sbe2a mutant leaves do not properly degrade starch during the night, resulting in hyperaccumulation. In mature sbe2a leaves, starch hyperaccumulation is greatest in visibly senescing regions but also observed in green tissue and is correlated to a drastic reduction in photosynthesis within the leaf. Starch granules from sbe2a leaves observed via scanning electron microscopy and transmission electron microscopy analyses are larger, irregular, and amorphous as compared with the highly regular, discoid starch granules observed in wild-type leaves. This appears to trigger premature senescence, as shown by an increased expression of genes encoding proteins known to be involved in senescence and programmed cell death processes. Together, these results indicate that SBEIIa is required for the proper diurnal cycling of transitory starch within the leaf and suggest that SBEIIa is necessary in producing an amylopectin structure amenable to degradation by starch metabolism enzymes.     

不同区域针叶年龄对红松叶性状及相关关系的影响

叶龄是植株个体发育过程中性状变异的重要影响因素, 不同叶龄下的性状变异可以体现植物针对环境变化做出的响应。该研究以东北地区4个不同区域阔叶红松(Pinus koraiensis)林中的成年红松为研究对象, 测定了4个区域红松的叶寿命和不同针叶年龄叶片的比叶面积(SLA)、叶片干物质含量(LDMC)、针叶密度(ND)以及针叶体积(NV), 探讨了红松叶形态性状间的相关性, 并检验了纬度和针叶年龄对这些相关关系的影响。结果显示: (1)不同纬度下红松叶寿命存在显著差异, 随纬度增加呈单峰模式。(2)针叶年龄在性状变异解释中占比最大(34.2%-80.1%), 对4个叶形态性状均存在显著影响。SLA、LDMC和ND在当年生叶片中均表现了比所有龄级下更大的可塑性, 仅NV在所有龄级下的可塑性大于当年生叶片。(3) 4个性状间的二元相关性显著, 且在不同纬度下多有体现, 但性状间的回归斜率随纬度变化趋势存在差异。(4)不同针叶年龄性状间的相关关系依然显著但变异规律不同, 如SLA与ND的回归斜率随针叶年龄增加而减小, ND与NV则相反。研究结果表明红松叶形态性状的变异和性状间的相关关系受纬度和针叶年龄的显著影响, 性状协同应对不同纬度梯度导致的环境变化以及不同针叶年龄下叶片的资源利用策略存在差异。     

ACC synthase expression regulates leaf performance and drought tolerance in maize

Ethylene regulates entry into several types of plant developmental cell death and senescence programs besides mediating plant responses to biotic and abiotic stress. The response of cereals to conditions of drought includes loss of leaf function and premature onset of senescence in older leaves. In this study, ACC synthase ( ACS ) mutants, affecting the first step in ethylene biosynthesis, were isolated in maize and their effect on leaf function examined. Loss of ZmACS6 expression resulted in delayed leaf senescence under normal growth conditions and inhibited drought-induced senescence. Zmacs6 leaves continued to be photosynthetically active under both conditions indicating that leaf function was maintained. The delayed senescence phenotype associated with loss of ZmACS6 expression was complemented by exogenous ACC. Surprisingly, elevated levels of foliar chlorophyll, Rubisco, and soluble protein as well as improved leaf performance was observed for all Zmasc6 leaves, including young and fully expanded leaves which were far from initiating senescence. These observations suggest that ethylene may serve to regulate leaf performance throughout its lifespan as well as to determine the onset of natural senescence and mediate drought-induced senescence.     

中国东部南北样带暖温带区栎属树种叶片形态性状对气候条件的响应

植物叶片的形态性状能够有效地反映生存环境的变化, 并且影响植物的基本行为和功能。该研究通过获取植物标本提供的叶片形态信息, 结合相关分析和标准化主轴分析, 探讨了南北样带暖温带区栎属(Quercus)树种叶片形态性状对气候条件的响应及适应策略。结果表明: 在南北样带暖温带区, 随着气候条件的变化, 栎属树种的叶片形态性状发生显著的变化。随着年平均气温的降低和年日照时数的增加, 栎属树种叶面积增加, 以利于吸收更多的光照辐射, 并增加叶片的边界层阻力, 减少叶片热量的散失; 而叶片分裂程度的增加不仅可以降低热量的散失, 也可以提高叶片液流的波动以增强叶片的生理活动; 叶脉密度随温度的升高、光照强度和降水量的增加而增加, 以响应叶片蒸腾作用的增强, 提高水分的运输能力和叶片的支撑能力。此外, 为适应南北样带暖温带区气候条件的变化, 栎属树种的叶片形成了一系列的形态性状组合, 随着叶面积的增加, 叶柄长度和叶片分裂程度逐渐增加, 而叶脉密度降低; 随着叶片倾向于向长条状发展, 叶柄长度和叶脉密度也随之增加。     

Accumulation of pathogenesis-related proteins in barley leaf intercellular spaces during leaf senescence

Accumulation of the pathogenesis-related (PR) proteins localised in intercellular spaces of barley primary leaves, chlorophyll content, structure of chloroplasts, and photosynthesis were examined during natural and in vitro induced leaf senescence (cultivation of whole plants in the dark or detached leaves under nutrient deficiency). Some of PR proteins accumulated during natural senescence, but their accumulation pattern was different from those of pathogen-induced as well as during in vitro-induced senescence, which indicate different molecular bases of these processes. Photosynthetic rate and chlorophyll content indicate that natural senescence of barley primary leaves began from 15th day after sowing. In 35-d-old first leaves, the chloroplasts showed typical characteristics of senescence as significant decrease of size, greater grana, and prominent plastoglobuli. The chloroplasts contained more grana under in vitro induced senescence and they had reduced length in the dark. Correspondingly, accumulation of PR proteins was detectable on about the 15th day but the content of some PR proteins increased in later stages of senescence. This revised version was published online in July 2006 with corrections to the Cover Date.