Activity and expression of polygalacturonase vary at different fruit ripening stages of sweet pepper cultivars

Activity and expression of polygalacturonase (PG), a hydrolytic enzyme involved in ultrastructural changes in the pericarp of sweet pepper (Capsicum annaum), were investigated at different ripening stages of the pepper cultivars Mandi and Talanduo. Molecular cloning of CaPG was carried out by constructing a cDNA library from three stages of fruit ripening. Morphological determination, PG assay, RT-PCR, and ultrastructural studies were used to quantify changes in CaPG gene expression in the pericarp from green, color change and fully ripened stages. We found that CaPG gene expression, PG activity and striking changes in the structure of the cell wall occurred with the transition of ripening stages. CaPG gene expression was high (obvious PCR products) in mature and ripened stages of both cultivars; however, the CaPG gene was not expressed in preclimacteric fruits or vegetative tissues. We conclude that developmental regulation of CaPG gene expression is instrumental for sweet pepper fruit ripening; its expression during development leads to dissolution of middle lamella and eventually disruption of the fully ripened cell wall.     

Peach fruit ripening: A proteomic comparative analysis of the mesocarp of two cultivars with different flesh firmness at two ripening stages

A proteomic analysis was conducted on peach fruit mesocarp in order to better elucidate the biochemical and physiological events which characterize the transition of fruit from the “unripe” to the “ripe” phase.The first goal of the present work was to set-up a protocol suitable for improving protein extraction from peach mesocarp. The use of freeze-dried powdered tissue, together with the addition of phenol prior to the extraction with an aqueous buffer, significantly increased the protein yield and the quality of 2-DE gels. The proteomic profiles of the mesocarp from peach fruit of a non-melting flesh (NMF; ‘Oro A’) and a melting flesh (MF; ‘Bolero’) cultivar, at “unripe” and “ripe” stages as defined by some parameters typical of ripening, were then analyzed.The comparative analysis of the 2-DE gels showed that in NMF and MF peaches the relative volumes of 53 protein spots significantly changed in relation to both the ripening stage (“unripe” versus “ripe”) and/or the genetic background of the cultivar (‘Oro A’ versus ‘Bolero’).Thirty out of the 53 differently abundant spots were identified by LC-ESI-MS/MS. The analysis revealed enzymes involved in primary metabolism (e.g. C-compounds, carbohydrates, organic acids and amino acids) and in ethylene biosynthesis as well as proteins involved in secondary metabolism and responses to stress.Among these, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) appeared to be one of the proteins with the largest change in relative abundance during the fruit transition from the pre-climacteric (“unripe”) to the climacteric (“ripe”) phase. Other proteins, such as S-adenosylmethionine synthetase and β-cyanoalanine synthase involved in ethylene metabolism, were also identified. Moreover, the changes in the relative abundances of a sucrose synthase and an α-amylase suggested differences between the two cultivars in the carbohydrate import activity of ripe fruit. The different accumulation of a few typical ROS-scavenger enzymes suggested that a higher oxidative stress occurred in MF with respect to NMF fruit. This result, together with data concerning the levels of total proteins and free amino acids and those regarding proteins involved in the maintenance of tissue integrity, was consistent with the hypothesis that the last phase of ripening in MF fruit is characterized by the appearance of a senescence status.The present study appears to define well some of the biochemical and physiological events that characterize the ripening of peach and, at the same time, provides interesting indications that could be employed in future marker assisted selection (MAS) programmes aimed to obtain MF fruits with higher ability to preserve tissue functionality maintaining for a longer time their organoleptic characteristics.     

AFLP analysis of genetic diversity within and among Coffea arabica cultivars

Genetic diversity of Coffea arabica cultivars was estimated using amplified fragment length polymorphism (AFLP) markers. Sixty one Coffea accessions composed of six arabica cultivars, including Typica, Bourbon, Catimor, Catuai, Caturra and Mokka Hybrid, plus two diploid Coffea species, were analyzed with six EcoRI- MseI primer combinations. A total of 274 informative AFLP markers were generated and scored as binary data. These data were analyzed using cluster methods in the software package NTSYSpc. The differences among cultivars at the DNA level were small, with an average genetic similarity of 0.933. Most accessions within a cultivar formed a cluster, although deviant samples occurred in five of the six cultivars examined due to residual heterozygosity from ancestral materials. Among the six cultivars fingerprinted, the highest level of genetic diversity was found within the cultivar Catimor, with an average genetic similarity of 0.880. The lowest level was found within Caturra accessions, with an average genetic similarity of 0.993. Diversity between C. arabica and two other Coffea species, Coffea canephora and Coffea liberica, was also estimated with average genetic similarities of 0.540 and 0.413, respectively, suggesting that C. canephora is more closely related to C. arabica than is C. liberica. The genetic variation among arabica cultivars was similar to the variation within cultivars, and no cultivar-specific DNA marker was detected. Although arabica cultivars appear to have a narrow genetic base, our results show that sufficient polymorphism can be found among some arabica cultivars with a genetic similarity as low as 0.767 for genetic/QTL mapping and breeding. The assessment of genetic diversity among arabica cultivars provided the necessary information to estimate the potential for using marker-assisted breeding for coffee improvement.     

Bee pollination and fruit set of Coffea arabica and C. canephora (Rubiaceae)

Self-sterile Coffea canephora and self-fertile C. arabica are important cash crops in many tropical countries. We examined the relative importance of insect, wind, and spontaneous self-pollination and the degree of self-fertility of these two coffee species in 24 agroforestry coffee fields in Indonesia. In both species, open pollination and cross pollination by hand led to the highest fruit set. Wind pollination (including self-pollination) led to 16% lower fruit set than open pollination in C. canephora and to 12.3% lower fruit set in C. arabica. Self-pollinated flowers and unmanipulated controls achieved an extremely low fruit set of 10% or less in the self-sterile species, and of 60% and 48%, respectively in the self-fertile species. These results constitute experimental evidence that cross pollination by bees causes a significant increase in fruit set of not only the self-sterile, but also the self-fertile coffee species. The practical implication is that coffee yield may be improved by managing fields for increased flower visitation by bees.     

Survival of salmonellae on and in tomato plants from the time of inoculation at flowering and early stages of fruit development through fruit ripening

The fate of salmonellae applied to tomato plants was investigated. Five Salmonella serotypes were used to inoculate tomato plants before and after fruits set, either by injecting stems with inoculum or brushing flowers with it. Ripe tomato fruits were subjected to microbiological analysis. Peptone wash water, homogenates of stem scar tissues, and homogenates of fruit pulp were serially diluted and plated on bismuth sulfite agar before and after enrichment. Presumptive Salmonella colonies were confirmed by serological tests, PCR assay using HILA2 primers, and enterobacterial repetitive intergenic consensus PCR. Of 30 tomatoes harvested from inoculated plants, 11 (37%) were positive for Salmonella. Of the Salmonella-positive tomatoes, 43 and 40%, respectively, were from plants receiving stem inoculation before and after flower set. Two of eight tomatoes produced from inoculated flowers contained Salmonella. Higher percentages of surface (82%) and stem scar tissue (73%) samples, compared to pulp of Salmonella-positive tomatoes (55%), harbored the pathogen. Of the five serotypes in the inoculum, Montevideo was the most persistent, being isolated from tomatoes 49 days after inoculation, and Poona was the most dominant, being present in 5 of 11 Salmonella-positive tomatoes. Results suggest that Salmonella cells survive in or on tomato fruits from the time of inoculation at flowering through fruit ripening. Tomato stems and flowers are possible sites at which Salmonella may attach and remain viable during fruit development, thus serving as routes or reservoirs for contaminating ripened fruit.     

Biochemical and genomic analysis of sucrose metabolism during coffee (Coffea arabica) fruit development

Sucrose metabolism and the role of sucrose synthase were investigated in the fruit tissues (pericarp, perisperm, and endosperm) of Coffea arabica during development. Acid invertase, sucrose phosphate synthase, and sucrose synthase activities were monitored and compared with the levels of sucrose and reducing sugars. Among these enzymes, sucrose synthase showed the highest activities during the last stage of endosperm and pericarp development and this activity paralleled closely the accumulation of sucrose in these tissues at this stage. Carbon partitioning in fruits was studied by pulse-chase experiments with (14)C-sugars and revealed high rates of sucrose turnover in perisperm and endosperm tissues. Additional feeding experiments with (14)CO(2) showed that leaf photosynthesis contributed more to seed development than the pericarp in terms of photosynthate supply to the endosperm. Sugar analysis, feeding experiments, and histological studies indicated that the perisperm plays an important role in this downloading process. It was observed that the perisperm presents a transient accumulation of starch which is degraded as the seed develops. Two full-length cDNAs (CaSUS1 and CaSUS2) and the complete gene sequence of the latter were also isolated. They encode sucrose synthase isoforms that are phylogenetically distinct, indicating their involvement in different physiological functions during cherry development. Contrasting expression patterns were observed for CaSUS1 and CaSUS2 in perisperm, endosperm, and pericarp tissues: CaSUS1 mRNAs accumulated mainly during the early development of perisperm and endosperm, as well as during pericarp growing phases, whereas those of CaSUS2 paralleled sucrose synthase activity in the last weeks of pericarp and endosperm development. Taken together, these results indicate that sucrose synthase plays an important role in sugar metabolism during sucrose accumulation in the coffee fruit.     

Identification of novel and conserved microRNAs in Coffea canephora and Coffea arabica

As microRNAs (miRNAs) are important regulators of many biological processes, a series of small RNAomes from plants have been produced in the last decade. However, miRNA data from several groups of plants are still lacking, including some economically important crops. Here microRNAs from Coffea canephora leaves were profiled and 58 unique sequences belonging to 33 families were found, including two novel microRNAs that have never been described before in plants. Some of the microRNA sequences were also identified in Coffea arabica that, together with C. canephora, correspond to the two major sources of coffee production in the world. The targets of almost all miRNAs were also predicted on coffee expressed sequences. This is the first report of novel miRNAs in the genus Coffea, and also the first in the plant order Gentianales. The data obtained establishes the basis for the understanding of the complex miRNA-target network on those two important crops.     

Genetic molecular analysis of Coffea arabica (Rubiaceae) hybrids using SRAP markers

In Coffea arabica (arabica coffee), the phenotypic as well as genetic variability has been found low because of the narrow genetic basis and self fertile nature of the species. Because of high similarity in phenotypic appearance among the majority of arabica collections, selection of parental lines for inter-varietals hybridization and identification of resultant hybrids at an early stage of plant growth is difficult. DNA markers are known to be reliable in identifying closely related cultivars and hybrids. Sequence Related Amplified Polymorphism (SRAP) is a new molecular marker technology developed based on PCR. In this paper, sixty arabica-hybrid progenies belonging to six crosses were analyzed using 31 highly polymorphic SRAP markers. The analysis revealed seven types of SRAP marker profiles which are useful in discriminating the parents and hybrids. The number of bands amplified per primer pair ranges from 6.13 to 8.58 with average number of seven bands. Among six hybrid combinations, percentage of bands shared between hybrids and their parents ranged from 66.29% to 85.71% with polymorphic bands varied from 27.64% to 60.0%. Percentage of hybrid specific fragments obtained in various hybrid combinations ranged from 0.71% to 10.86% and ascribed to the consequence of meiotic recombination. Based on the similarity index calculation, it was observed that F1 hybrids share maximum number of bands with the female parent compared to male parent. The results obtained in the present study revealed the effectiveness of SRAP technique in cultivar identification and hybrid analysis in this coffee species.     

Physiological and chemical indicators for early and late stages of sepsis in conscious rats

Endotoxin shock is a major cause of death in patients with septicemia. Endotoxin induces nitric oxide (NO) production and causes tissue damage. In addition, the release of oxygen free radicals has also been observed in endotoxin shock and was found to be responsible for the occurrence of multiple organ failure. The purpose of the present study was to evaluate suitable indicators for early and late stages of endotoxin shock. The experiments were designed to induce endotoxin shock in conscious rats by means of anEscherichia coli lipopolysaccharide (LPS) injection. Arterial pressure (AP) and heart rate (HR) were continuously monitored for 72 h after LPS administration. The maximal decrease in AP and increase in HR and nitrate/nitrite level occurred at 9–12 h following LPS administration. The white blood cell (WBC) count had decreased at 3 h. Hydroxyl radical (methyl guanidine, MG) decreased rapidly after LPS administration. Plasma levels of blood urea nitrogen (BUN), creatinine (Cr), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), and glutamic oxaloacetic transaminase increased before the rise of amylase. Our results suggest that changes in AP, HR, WBC, free radicals, and chemical substances (BUN, Cr) can possibly serve as approximate indicators for the early stage of endotoxin shock. Severe multiple organ damage may be caused by amylase release in the late stage of endotoxin shock.     

田间不同水肥管理下小粒咖啡的生长和光合特性

通过云南5年生田间小粒咖啡(Coffea arabica)进行2种施肥(低肥和高肥)和在干季秸秆覆盖、滴灌、秸秆覆盖+滴灌、对照4种水分处理对植株生长和叶片光合特性影响的测定研究．结果表明,小粒咖啡一年生长周期中最高峰期在雨季始期,次高峰期在雨季中期．水分处理对生长高峰期株高和分枝长度的相对生长率没有显著作用,高施肥量则加大了其相对生长率．干季水分处理提高了叶片的Pn、gs、Tr和WUE,而叶绿素的荧光特征没有受到影响．在湿季,高施肥量使叶片含氮量Pn增加,对gs和Tr的影响较小,从而导致WUE提高,高施肥量显著减小日间光抑制程度,加大了光合机构的实际光化学效率和热耗散能力,提高了对强光环境的适应性,研究表明,小粒咖啡需要高养分的投入和良好的水分管理,湿季是小粒咖啡进行光合和生长的最优季节,干季田间秸秆覆盖+滴灌的效果较好,滴灌和秸秆覆盖的效果相近。     

不同氮效率水稻生育后期根表和根际土壤硝化特征

通过田间试验研究了不同氮效率粳稻品种4007(氮高效)和Elio(氮低效)生育后期在N0(0 kgN hm-2)、N180(180 kgN hm-2)和N300(300 kgN hm-2)水平下根表、根际和土体土壤pH值、铵态氮(NH+4-N)和硝态氮(NO-3-N)含量、硝化强度和氨氧化细菌(AOB)数量.结果表明无论是齐穗期、灌浆期还是成熟期,根表土壤pH值均显著低于根际和土体土壤.土壤pH值范围在5.95至6.84之间变化.土壤NH+4-N含量随水稻生长显著下降,且随施氮量增加而显著增加.根表土壤NH+4-N有明显亏缺区,且随距水稻根表距离增加,NH+4-N含量逐渐升高.土壤NO-3-N含量随水稻生长显著增加,施氮处理均显著高于不施氮处理,但N180和N300处理差异不显著.NO-3-N含量表现为根际>土体>根表.水稻根表和根际土壤硝化强度随水稻生长显著下降,而土体土壤硝化强度随时间延长小幅增加.施氮显著提高4007水稻根表土壤在齐穗和收获期硝化强度以及Elio在齐穗期根际硝化强度,但在施氮处理N180和N300中无显著差异.在整个采样期间,土壤硝化强度均表现为根际>根表>土体.水稻根表和根际AOB数量随水稻生长而显著降低,而土体土壤AOB数量无显著变化.例如,根表土壤AOB数量在齐穗期、灌浆期和收获期分别为16.7×105、8.77×105个g-1 dry soil和8.01×105个g-1 dry soil.根表和根际土壤AOB数量无显著差异,但二者显著高于土体土壤AOB数量.就两个氮效率水稻品种而言,土壤pH值基本无差异.4007土壤NH+4-N含量均显著高于Elio.在齐穗期水稻根表、根际和土体土壤NO-3-N含量在N180水平下均表现为Elio显著高于4007.而在灌浆期和收获期,水稻根表、根际和土体土壤则表现为4007显著高于Elio.在所有采样期,两个水稻品种土体土壤硝化强度和AOB数量在3个施氮量下均无显著差异.Elio根表和根际土壤硝化强度和AOB数量在水稻灌浆期之前一直显著高于4007,而在灌浆期之后则显著低于4007,且最终产量和氮素利用率(NUE)显著低于4007,这可能是由于4007灌浆期后硝化作用强,根际产生的NO-3-N含量高,从而4007根吸收NO-3-N的量也高造成的.因此水稻灌浆期和收获期根表和根际硝化作用以及AOB与水稻高产及氮素高效利用密切相关.     

Physiological and morphological changes during early and later stages of fruit growth in Capsicum annuum

Fruit‐set involves a series of physiological and morphological changes that are well described for tomato and Arabidopsis, but largely unknown for sweet pepper (Capsicum annuum). The aim of this paper is to investigate whether mechanisms of fruit‐set observed in Arabidopsis and tomato are also applicable to C. annuum. To do this, we accurately timed the physiological and morphological changes in a post‐pollinated and un‐pollinated ovary. A vascular connection between ovule and replum was observed in fertilized ovaries that undergo fruit development, and this connection was absent in unfertilized ovaries that abort. This indicates that vascular connection between ovule and replum is an early indicator for successful fruit development after pollination and fertilization. Evaluation of histological changes in the carpel of a fertilized and unfertilized ovary indicated that increase in cell number and cell diameter both contribute to early fruit growth. Cell division contributes more during early fruit growth while cell expansion contributes more at later stages of fruit growth in C. annuum. The simultaneous occurrence of a peak in auxin concentration and a strong increase in cell diameter in the carpel of seeded fruits suggest that indole‐3‐acetic acid stimulates a major increase in cell diameter at later stages of fruit growth. The series of physiological and morphological events observed during fruit‐set in C. annuum are similar to what has been reported for tomato and Arabidopsis. This indicates that tomato and Arabidopsis are suitable model plants to understand details of fruit‐set mechanisms in C. annuum.     

Auxin-regulated polypeptide changes at different stages of strawberry fruit development

The pattern of polypeptides at different stages of strawberry (Fragaria ananassa Duch. cv Ozark Beauty) fruit development was studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An 81,000-dalton polypeptide appeared between 5 and 10 days after pollination. Polypeptides with molecular weights of 76,000 and 37,000 daltons were formed after 10 days. The control exerted by auxin in the stage-specific formation of polypeptides was investigated by stopping fruit growth after removing the achenes and reinitiating fruit growth by the application of a synthetic auxin, α-naphthaleneacetic acid (NAA). When the achenes were removed from the 5- and 10-day-old fruits, the fruits failed to grow, the 81,000 dalton polypeptide was not formed between 5 and 10 days, and the 76,000- and 37,000-dalton polypeptides were not formed between 10 and 20 days. Application of NAA to fruits deprived of auxin by removal of achenes resulted in the resumption of growth and also in the appearance of these polypeptides. Removal of achenes of the 5- or 10-day-old fruits and growing them without auxin resulted in the formation of 52,000- and 57,000-dalton polypeptides. These two polypeptides were not formed when NAA was applied to fruits after removal of achenes. Supply of NAA to auxin-deprived fruits 5 days after removal of achenes resulted in resumption of growth and also in the disappearance of these two polypeptides, pointing out their possible relation to the inhibition of fruit growth.     

Biochemical and molecular characterization and expression of the 11S-type storage protein from Coffea arabica endosperm

In order to define better the endosperm protein content of commercial coffee species Coffea arabica (Arabica) and C. canephora (Robusta), the principal storage protein of coffee grains has been analysed by 2-dimensional electrophoresis (2DE) and amino acid microsequencing. The most abundant polypeptide spots observed on mature coffee grain 2DE profiles were found to be subunits of the same protein, which exists as multiple isoforms with varying pIs. Strong sequence similaritywas found to the 11S family of plant storage proteins. The structure is typical of the 11S type, which occurs as a precursor of 55 kDa, and is observed under denaturing and reducing conditions on 2DE profiles in the form of cleavage products at approximately 20 kDa (β arms) and 32 kDa (α arms). Differences between Arabica and Robusta 2DE profiles indicate a secondary 11S protein family in some varieties of the latter. The existence of multiple pI forms may indicate that a multigene family encodes for these proteins. We estimate that the protein accounts for approximately 45 % of total grain protein. A cloned full-length cDNA of 1 706 bp coding for one of the isoforms is described and discussed in relation to other coffee storage protein sequences.     

刺芹侧耳不同发育时期的转录组差异分析

为了探讨刺芹侧耳子实体生长发育时期的基因表达变化,本文利用高通量测序技术对刺芹侧耳不同发育时期（菌丝期、原基期、子实体时期）进行RNA-Seq分析,在转录水平上解析差异表达基因在刺芹侧耳生长发育过程中的作用和功能。KEGG功能富集显示,菌丝期差异表达基因主要富集在碳代谢和氨基酸代谢中,其中三羧酸循环中编码柠檬酸合酶、乌头酸水合酶、异柠檬酸脱氢酶、琥珀酰辅酶A合成酶、琥珀酸脱氢酶、苹果酸脱氢酶的基因表达量均上调,说明碳代谢和氨基酸代谢是菌丝时期的主要能量来源;原基期上调的差异表达基因主要富集在脂肪酸代谢,其中RT-PCR定量结果显示原基期编码脂肪酸合酶的基因和编码脂酰辅酶A合成酶的基因下调,编码超氧化物酶的基因和编码过氧化氢酶的基因上调,表明脂肪酸代谢和抗氧化酶对刺芹侧耳原基期维持机体的稳定和生物应激方面起着重要作用。子实体时期上调的差异表达基因主要富集在剪接体、类固醇的生物合成以及AMPK信号通路中,说明环境因子对子实体时期有一定的影响。