Enhancement of NADP-malic enzyme in transgenic rice induced the accumulation of reactive oxygen species

It had been demonstrated that the photosynthetic photodamage, such as photoinhibition and photooxidation, was enhanced in transgenic rice plants overexpressing NADP-malic enzyme (ME). However, its physiological base has not been investigated. In order to elucidate the physiological elements contributed to the enhancement of photodamage in NADP-ME transgenic rice plants, some physiological indices related to reactive oxygen species (ROS) accumulation were studied using the T₁ progeny of transgenic rice plants. The results showed that more ROS such as O ₂ ^−. and H₂O₂ were accumulated in transgenic rice plants, which enhanced photooxidation, while the accumulation of malondialdehyde in transgenic rice plants was not evident as compared with the wild-type plants. The measurement of NADPH/NADP ratios in leaves showed that transgenic rice plants had a higher ratio than untransformed rice plants. Based on these data, we speculated that overexpression of NADP-ME led to the deficiency of NADP and overreduction of photosystem I, which induced the accumulation of ROS in the transgenic rice plants, and just ROS were accounted for plant sensitivity to photooxidation. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 3, pp. 364–370. The text was submitted by the author in English.     

Identification of early senescence-associated genes in rice flag leaves

Leaf senescence is one of the key stages of plant leaf development. It is a highly complex but ordered process involving expression of large scale senescence associated genes, and its molecular mechanisms still remain unclear. By using suppression subtractive hybridization, 815 ESTs that are up-regulated at the onset of rice flag leaf senescence have been isolated. A total of 533 unigenes have been confirmed by macroarray detection and sequencing. 183 of these unigenes have GO annotations, involved in macromolecule metabolism, protein biosynthesis regulation, energy metabolism, gene expression regulations, detoxification, pathogenicity and stress, cytoskeleton organization and flower development. Another 121 unigenes co-localized with previously reported known stay-green QTLS. RT-PCR analysis on the other novel genes indicated that they can be up-regulated in natural early senescence and induced by hormone. Our results indicate that senescence is closely related to various metabolic pathways, thus providing new insight into the onset of leaf senescence mechanism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Phytohormones and microRNAs as sensors and regulators of leaf senescence: Assigning macro roles to small molecules

Ageing or senescence is an intricate and highly synchronized developmental phase in the life of plant parts including leaf. Senescence not only means death of a plant part, but during this process, different macromolecules undergo degradation and the resulting components are transported to other parts of the plant. During the period from when a leaf is young and green to the stage when it senesces, a multitude of factors such as hormones, environmental factors and senescence associated genes (SAGs) are involved. Plant hormones including salicylic acid, abscisic acid, jasmonic acid and ethylene advance leaf senescence, whereas others like cytokinins, gibberellins, and auxins delay this process. The environmental factors which generally affect plant development and growth, can hasten senescence, the examples being nutrient dearth, water stress, pathogen attack, radiations, high temperature and light intensity, waterlogging, and air, water or soil contamination. Other important influences include carbohydrate accumulation and high carbon/nitrogen level. To date, although several genes involved in this complex process have been identified, still not much information exists in the literature on the signalling mechanism of leaf senescence. Now, the Arabidopsis mutants have paved our way and opened new vistas to elucidate the signalling mechanism of leaf senescence for which various mutants are being utilized. Recent studies demonstrating the role of microRNAs in leaf senescence have reinforced our knowledge of this intricate process. This review provides a comprehensive and critical analysis of the information gained particularly on the roles of several plant growth regulators and microRNAs in regulation of leaf senescence.     

SUB1A‐dependent and ‐independent mechanisms are involved in the flooding tolerance of wild rice species

Crop tolerance to flooding is an important agronomic trait. Although rice (Oryza sativa) is considered a flood‐tolerant crop, only limited cultivars display tolerance to prolonged submergence, which is largely attributed to the presence of the SUB1A gene. Wild Oryza species have the potential to unveil adaptive mechanisms and shed light on the basis of submergence tolerance traits. In this study, we screened 109 Oryza genotypes belonging to different rice genome groups for flooding tolerance. Oryza nivara and Oryza rufipogon accessions, belonging to the A‐genome group, together with Oryza sativa, showed a wide range of submergence responses, and the tolerance‐related SUB1A‐1 and the intolerance‐related SUB1A‐2 alleles were found in tolerant and sensitive accessions, respectively. Flooding‐tolerant accessions of Oryza rhizomatis and Oryza eichingeri, belonging to the C‐genome group, were also identified. Interestingly, SUB1A was absent in these species, which possess a SUB1 orthologue with high similarity to O. sativa SUB1C. The expression patterns of submergence‐induced genes in these rice genotypes indicated limited induction of anaerobic genes, with classical anaerobic proteins poorly induced in O. rhizomatis under submergence. The results indicated that SUB1A‐1 is not essential to confer submergence tolerance in the wild rice genotypes belonging to the C‐genome group, which show instead a SUB1A‐independent response to submergence.     

Cytogenetic analysis of human cells reveals specific patterns of DNA damage in replicative and oncogene‐induced senescence

Senescence is thought to be triggered by DNA damage, usually indirectly assessed as activation of the DNA damage response (DDR), but direct surveys of genetic damage are lacking. Here, we mitotically reactivate senescent human fibroblasts to evaluate their cytogenetic damage. We show that replicative senescence is generally characterized by telomeric fusions. However, both telomeric and extratelomeric aberrations are prevented by hTERT, indicating that even non‐telomeric damage descends from the lack of telomerase. Compared with replicative senescent cells, oncogene‐induced senescent fibroblasts display significantly higher levels of DNA damage, depicting how oncogene activation can catalyze the generation of further, potentially tumorigenic, genetic damage.     

Persistent DNA damage‐induced premature senescence alters the functional features of human bone marrow mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout life to ensure tissue homeostasis and repair. For this reason, they must be equipped with DNA damage responses able to maintain genomic integrity while ensuring their lifelong persistence. Evaluation of hMSC response to genotoxic insults is of great interest considering both their therapeutic potential and their physiological functions. This study aimed to investigate the response of human bone marrow MSCs to the genotoxic agent Actinomycin D (ActD), a well‐known anti‐tumour drug. We report that hMSCs react by undergoing premature senescence driven by a persistent DNA damage response activation, as hallmarked by inhibition of DNA synthesis, p21 and p16 protein expression, marked Senescent Associated β‐galactosidase activity and enlarged γH2AX foci co‐localizing with 53BP1 protein. Senescent hMSCs overexpress several senescence‐associated secretory phenotype (SASP) genes and promote motility of lung tumour and osteosarcoma cell lines in vitro. Our findings disclose a multifaceted consequence of ActD treatment on hMSCs that on the one hand helps to preserve this stem cell pool and prevents damaged cells from undergoing neoplastic transformation, and on the other hand alters their functional effects on the surrounding tissue microenvironment in a way that might worsen their tumour‐promoting behaviour.     

过表达大豆类受体蛋白激酶基因RLPK2促进转基因拟南芥叶片的衰老北大核心CSCD

大豆RLPK2基因(GenBank登录号:AY687391)是一个编码N-末端富含亮氨酸重复序列的类受体蛋白激酶基因。为分析大豆RLPK2基因的功能,该研究以野生型拟南芥和大豆RLPK2基因过表达拟南芥植株为材料,通过农杆菌介导法转化野生型拟南芥,构建了大豆RLPK2基因过表达载体,分析了叶片衰老过程中叶绿素荧光参数、抗氧化酶活性及衰老相关基因表达量的变化。结果表明:(1)无论是野生型还是转基因拟南芥,随着叶片衰老进程的进行,光系统Ⅱ(PSⅡ)的最大光化学效率(F_(v)/F_(m))、PSⅡ实际光化学效率(Φ_(PSⅡ))、光化学淬灭系数(qP)和光合电子传递速率(ETR)均呈下降趋势,但后者下降趋势更明显;(2)激发压(1-qP)在叶片衰老前期的变化较为平稳,后期则急剧增加,且转基因型比野生型拟南芥增加更明显;(3)在叶片衰老的各个时期,转基因拟南芥叶片丙二醛(MDA)含量均显著高于野生型,而超氧化物岐化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)活性均显著低于野生型;(4)实时荧光定量PCR检测结果表明,RLPK2转基因拟南芥中衰老标志基因ATSAG12,衰老关键转录因子ATNAP、ATWRKY6和叶绿素降解关键酶编码基因ATACD1表达量显著上调。综上认为,大豆类受体蛋白激酶基因RLPK2参与调控植物叶片衰老进程,其表达对叶片衰老具有促进作用。     

水稻长穗颈基因eui紧密连锁SSR标记获得

02428h是从半矮秆材料02428体细胞培养后代中发现的隐性高秆突变体, 其株高性状由1对长穗颈基因eui和1对半矮秆基因sd-1共同控制。以02428h与半矮秆材料南京11杂交的F₂为作图群体, 利用Gramene公布的SSR标记和根据NCBI中的BAC序列自己新开发的SSR标记, 将eui基因定位在第5染色体上的RM3673和RM0012之间, 两侧遗传距离分别为0.3 cM和1.0 cM, 为该基因的分子标记辅助选择奠定了基础。     

New changes in the plasma‐membrane‐associated proteome of rice roots under salt stress

To gain a better understanding of salt stress responses in plants, we used a proteomic approach to investigate changes in rice (Oryza sativa) root plasma‐membrane‐associated proteins following treatment with 150 mmol/L NaCl. With or without a 48 h salt treatment, plasma membrane fractions from root tip cells of a salt‐sensitive rice cultivar, Wuyunjing 8, were purified by PEG aqueous two‐phase partitioning, and plasma‐membrane‐associated proteins were separated by IEF/SDS‐PAGE using an optimized rehydration buffer. Comparative analysis of three independent biological replicates revealed that the expressions of 18 proteins changed by more than 1.5‐fold in response to salt stress. Of these proteins, nine were up‐regulated and nine were down‐regulated. MS analysis indicated that most of these membrane‐associated proteins are involved in important physiological processes such as membrane stabilization, ion homeostasis, and signal transduction. In addition, a new leucine‐rich‐repeat type receptor‐like protein kinase, OsRPK1, was identified as a salt‐responding protein. Immuno‐blots indicated that OsRPK1 is also induced by cold, drought, and abscisic acid. Using immuno‐histochemical techniques, we determined that the expression of OsRPK1 was localized in the plasma membrane of cortex cells in roots. The results suggest that different rice cultivars might have different salt stress response mechanisms.     

A genome‐wide survey reveals abundant rice blast R genes in resistant cultivars

Plant resistance genes (R genes) harbor tremendous allelic diversity, constituting a robust immune system effective against microbial pathogens. Nevertheless, few functional R genes have been identified for even the best‐studied pathosystems. Does this limited repertoire reflect specificity, with most R genes having been defeated by former pests, or do plants harbor a rich diversity of functional R genes, the composite behavior of which is yet to be characterized? Here, we survey 332 NBS‐LRR genes cloned from five resistant Oryza sativa (rice) cultivars for their ability to confer recognition of 12 rice blast isolates when transformed into susceptible cultivars. Our survey reveals that 48.5% of the 132 NBS‐LRR loci tested contain functional rice blast R genes, with most R genes deriving from multi‐copy clades containing especially diversified loci. Each R gene recognized, on average, 2.42 of the 12 isolates screened. The abundant R genes identified in resistant genomes provide extraordinary redundancy in the ability of host genotypes to recognize particular isolates. If the same is true for other pathogens, many extant NBS‐LRR genes retain functionality. Our success at identifying rice blast R genes also validates a highly efficient cloning and screening strategy.     

Temporal and spatial regulation of DROOPING LEAF gene expression that promotes midrib formation in rice

Genes involved in the differentiation and development of tissues and organs are temporally and spatially regulated in plant development. The DROOPING LEAF (DL) gene, a member of the YABBY gene family, promotes midrib formation in the leaf and carpel specification in the flower. Consistent with these functions, DL is initially expressed in the central region of the leaf primordia (presumptive midrib) and in the presumptive carpel primordia in the meristem. To understand the regulatory mechanism underlying DL expression, we tried to identify cis-regulatory regions required for temporal and spatial expression of this gene. We found that the cis region responsible for the presumptive midrib-specific expression in the leaf primordia is located in intron 2. Next, we confined the region to a sequence of about 200bp, which corresponds to a conserved non-coding sequence (CNS) identified by phylogenetic footprinting. In addition, a sequence termed DG1, incorporating a 5' upstream region of about 7.4kb, and introns 1 and 2, was shown to be sufficient to induce DL in the presumptive midrib, and to suppress it in other regions in the leaf primordia. By contrast, the regulatory region required for carpel-specific expression was not included in the DG1 sequence. We modified Oryza sativa (rice) plant architecture by expressing an activated version of DL (DL-VP16) in a precise manner using the DG1 sequence: the resulting transgenic plant produced a midrib in the distal region of the leaf blade, where there is no midrib in wild type, and formed more upright leaves compared with the wild type.     

OsMOGS is required for N‐glycan formation and auxin‐mediated root development in rice (Oryza sativa L.)

N‐glycosylation is a major modification of glycoproteins in eukaryotic cells. In Arabidopsis, great progress has been made in functional analysis of N‐glycan production, however there are few studies in monocotyledons. Here, we characterized a rice (Oryza sativa L.) osmogs mutant with shortened roots and isolated a gene that coded a putative mannosyl‐oligosaccharide glucosidase (OsMOGS), an ortholog of α‐glucosidase I in Arabidopsis, which trims the terminal glucosyl residue of the oligosaccharide chain of nascent peptides in the endoplasmic reticulum (ER). OsMOGS is strongly expressed in rapidly cell‐dividing tissues and OsMOGS protein is localized in the ER. Mutation of OsMOGS entirely blocked N‐glycan maturation and inhibited high‐mannose N‐glycan formation. The osmogs mutant exhibited severe defects in root cell division and elongation, resulting in a short‐root phenotype. In addition, osmogs plants had impaired root hair formation and elongation, and reduced root epidemic cell wall thickness due to decreased cellulose synthesis. Further analysis showed that auxin content and polar transport in osmogs roots were reduced due to incomplete N‐glycosylation of the B subfamily of ATP‐binding cassette transporter proteins (ABCBs). Our results demonstrate that involvement of OsMOGS in N‐glycan formation is required for auxin‐mediated root development in rice.     

Tissue‐specific and light‐dependent regulation of phytochrome gene expression in rice

Phytochromes are red‐ and far red light photoreceptors in higher plants. Rice (Oryza sativa L.) has three phytochromes (phyA, phyB and phyC), which play distinct as well as cooperative roles in light perception. To gain a better understanding of individual phytochrome functions in rice, expression patterns of three phytochrome genes were characterized using promoter‐GUS fusion constructs. The phytochrome genes PHYA and PHYB showed distinct patterns of tissue‐ and developmental stage‐specific expression in rice. The PHYA promoter‐GUS was expressed in all leaf tissues in etiolated seedlings, while its expression was restricted to vascular bundles in expanded leaves of light‐grown seedlings. These observations suggest that light represses the expression of the PHYA gene in all cells except vascular bundle cells in rice seedlings. Red light was effective, but far red light was ineffective in gene repression, and red light‐induced repression was not observed in phyB mutants. These results indicate that phyB is involved in light‐dependent and tissue‐specific repression of the PHYA gene in rice.