Gang Pan

The chloroplast ribosomal protein L21 gene is essential for plastid development and embryogenesis in Arabidopsis

Embryogenesis in higher plants is controlled by a complex gene network. Identification and characterization of genes essential for embryogenesis will provide insights into the early events in embryo development. In this study, a novel mutant with aborted seed development (asd) was identified in Arabidopsis. The asd mutant produced about 25% of albino seeds at the early stage of silique development. The segregation of normal and albino seeds was inherited as a single recessive embryo-lethal trait. The gene disrupted in the asd mutant was isolated through map-based cloning. The mutated gene contains a single base change (A to C) in the coding region of RPL21C (At1g35680) that is predicted to encode the chloroplast 50S ribosomal protein L21. Allele test with other two T-DNA insertion lines in RPL21C and a complementation test demonstrated that the mutation in RPL21C was responsible for the asd phenotype. RPL21C exhibits higher expression in leaves and flowers compared with expression levels in roots and developing seeds. The RPL21C–GFP fusion protein was localized in chloroplasts. Cytological observations showed that the asd embryo development was arrested at the globular stage. There were no plastids with normal thylakoids and as a result no normal chloroplasts formed in mutant cells, indicating an indispensable role of the ASD gene in chloroplasts biogenesis. Our studies suggest that the chloroplast ribosomal protein L21 gene is required for chloroplast development and embryogenesis in Arabidopsis.

Analyses of two rice (Oryza sativa) cyclin-dependent kinase inhibitors and effects of transgenic expression of OsiICK6 on plant growth and development

BACKGROUND AND AIMS Plants have a family of proteins referred to as ICKs (inhibitors of cyclin-dependent kinase, CDK) or KRPs (Kip-related proteins) that function to regulate the activities of CDK. Knowledge of these plant CDK inhibitors has been gained mostly from studies of selected members in dicotyledonous plants, particularly Arabidopsis. Much remains to be learned regarding the differences among various members of the ICK/KRP family, and regarding the function and regulation of these proteins in monocotyledonous plants. METHODS We analysed ICK-related sequences in the rice (Orysa sativa L. subsp. indica) genome and determined that there are six members with the conserved C-terminal signature region for ICK/KRP proteins. They are referred to as OsiICKs and further analyses were performed. The interactions with CDKs and cyclins were determined by a yeast two-hybrid assay, and cellular localization by fusion with the enhanced green fluorescence protein (EGFP). The expression of OsiICK6 in different tissues and in response to several treatments was analysed by reverse transcriptase-mediated polymerase chain reaction (RT-PCR) and real-time PCR. Furthermore, OsiICK6 was over-expressed in transgenic rice plants and significant phenotypes were observed. KEY RESULTS AND CONCLUSIONS Based on putative protein sequences, the six OsiICKs are grouped into two classes, with OsiICK1 and OsiICK6 in each of the two classes, respectively. Results showed that OsiICK1 and OsiICK6 interacted with OsCYCD, but differed in their interactions with CDKA. Both EGFP:OsiICK1 and EGFP:OsiICK6 were localized in the nucleus. Whereas EGFP:OsiICK6 showed a punctuate subnuclear distribution, OsiICK1 had a homogeneous pattern. Over-expression of OsiICK6 resulted in multiple phenotypic effects on plant growth, morphology, pollen viability and seed setting. In OsiICK6-over-expressing plants, leaves rolled toward the abaxial side, suggesting that cell proliferation is critical in maintaining an even growth along the dorsal-ventral plane of leaf blades.

Transcriptional profile of genes involved in ascorbate glutathione cycle in senescing leaves for an early senescence leaf (esl) rice mutant.

To clarify the complex relationship between ascorbate-glutathione (AsA-GSH) cycle and H2O2-induced leaf senescence, the genotype-dependent difference in some senescence-related physiological parameters and the transcript levels and the temporal patterns of genes involved in the AsA-GSH cycle during leaf senescence were investigated using two rice genotypes, namely, the early senescence leaf (esl) mutant and its wild type. Meanwhile, the triggering effect of exogenous H2O2 on the expression of OsAPX genes was examined using detached leaves. The results showed that the esl mutant had higher H2O2 level than its wild type at the initial stage of leaf senescence. At transcriptional level, the association of expression of various genes involved in the AsA-GSH cycle with leaf senescence was isoform dependent. For OsAPXs, the transcripts of two cytosolic OsAPX genes (OsAPX1 and OsAPX2), thylakoid-bound OsAPX8, chloroplastic OsAPX7 and peroxisomal OsAPX4 exhibited remarkable genotype-dependent variation in their expression levels and temporal patterns during leaf senescence, there were significantly increasing transcripts of OsAXP1 and OsAPX7, severely repressed transcripts of OsAPX4 and OsAPX8 for the esl rice at the initial leaf senescence. In contrast, the repressing transcript of OsAPX8 was highly sensitive to the increasing H2O2 level in the senescing rice leaves, while higher H2O2 concentration resulted in the enhancing transcripts of two cytosolic OsAPX genes, OsAPX7 transcript was greatly variable with different H2O2 concentrations and incubating duration, suggesting that the different OsAPXs isoforms played a complementary role in perceiving and scavenging H2O2 accumulation at various H2O2 concentrations during leaf senescence. Higher H2O2 level, increased AsA level, higher activities of APX and glutathione reductase (GR), and relatively stable GSH content during the entire sampling period in the leaves of esl mutant implied that a close interrelationship existed between AsA level and APX activity in the ongoing senescence of rice leaves. The GSH supply in rice leaves was not the limiting factor for the efficient maintenance of AsA-GSH cycle, despite the senescence-related change in GR activity between the two rice genotypes.

Senescence-specific change in ROS scavenging enzyme activities and regulation of various SOD isozymes to ROS levels in psf mutant rice leaves

To clarify the interaction between different antioxidant enzymes for monitoring oxidative stress and ROS burst in rice senescent leaves, we investigated the genotype-dependent alteration in temporal patterns of the O2•- production rate, H2O2 content, and ROS-scavenging enzyme activities during leaf senescence in two rice genotypes, namely, the premature senescence of flag leaf (psf) mutant and its wild type. Results showed that the psf mutant differed obviously from its wild type in leaf O2•- generation rate and H2O2 content accumulation, and the decreased activities of SOD, CAT, and APX in the psf leaves were strongly responsible for the increased ROS level and the accelerated leaf senescence. By contrast, the increase in POD activity was positively correlated with the senescence-related enhancement in O2•- generation in rice leaves. Among various SOD isoforms, Mn-SOD responded sensitively to the increasing O2•- generation rate, whereas Cu/Zn-SOD remained stable with the progression of leaf senescence. These findings suggest that the senescence-related decline in total SOD activity was mostly attributable to the downregulation of both the translation and transcription of Mn-SOD isoform. This occurrence finally resulted in the collapse of SOD defense system and accelerated leaf senescence for the psf mutant. Furthermore, we presented the possible contribution of several Cu/Zn-SOD expression patterns to the senescence-related O2•- detoxification in different cell compartments.

Non-target site mechanism of metribuzin tolerance in induced tolerant mutants of narrow-leafed lupin (Lupinus angustifolius L.)

Abstract. Narrow-leafed lupin (Lupinus angustifolius L.) is an important grain legume crop in Australia. Metribuzin is an important herbicide used to control weeds in lupin crops. This study investigated metribuzin tolerance mechanism in narrow-leafed lupin by comparing two induced mutants (Tanjil-AZ-33 and Tanjil-AZ-55) of higher metribuzin tolerance with the susceptible wild type. Sequencing of the highly conserved region of the chloroplast psbA gene (target site) revealed that the sequences of the wild type and the mutants were identical and therefore metribuzin tolerance is not target site based. Photosynthetic activity was measured and the leaf photosynthesis of the two tolerant mutants was initially inhibited after metribuzin treatment, but recovered within 2.5 days whereas that of the susceptible plants remained inhibited. The photosynthetic measurements confirmed the target site chloroplast was susceptible and the tolerance mechanism is non-target site based. Investigation with known cytochrome P450 monooxygenase inhibitors (omethoate, malathion and phorate) showed that tolerance could be reversed in both mutants, indicating the tolerance mechanism in two tolerant mutants may involve cytochrome P450 enzymes. Interestingly, the inhibitor tridiphane reversed metribuzin tolerance of only one of the two tolerant mutants, indicating diversity in metribuzin tolerance mechanisms in narrow-leafed lupin. These results signify that further investigation of metribuzin metabolism in these plants is warranted. In conclusion, metribuzin tolerance mechanism in lupin mutants is non-target site based, likely involving P450-mediated metribuzin metabolism.

A single cytosine deletion in the OsPLS1 gene encoding vacuolar-type H+-ATPase subunit A1 leads to premature leaf senescence and seed dormancy in rice.

Highlight The OsPLS1 locus that encodes VHA-A1 in rice, was identified by map-based cloning. OsPLS1/VHA-A1 is involved in premature leaf senescence and seed dormancy.

Effect of high temperature on the expressions of genes encoding starch synthesis enzymes in developing rice endosperms

Abstract High temperature is the major environmental factor affecting grain starch properties of cooking rice cultivars. In this study, two non-waxy indica rice genotypes, cv . 9311 and its mutant with extremely high amylose phenotype (9311 eha ) were used to study the differential expressions of genes in starch synthesis and their responses to high temperature (HT). Significant increase in apparent amylose content and hot-water-soluble starch content in mutant 9311 eha were genetically caused by a substitution from AGTTATA to AGGTATA at the leader intron 5′ splice site in Wx gene. This mutation resulted in different mRNA transcript levels, mRNA splicing efficiencies and protein levels of Wx between the two rice genotypes, which also lead to the genotype-dependent alteration in the temporal pattern of Wx transcription and granule-bound starch synthase (GBSS) activity in response to HT. However, changes in the activities of other starch synthesizing enzymes and their expressions of distinct isoform genes were not significant with the Wx gene mutation, thus only minor difference in the particle size of starch granule, chain-length distribution and gelatinization enthalpy were found between the two genotypes. The temporal-specific expression of multiple isoform genes responsive to different temperature regiments indicated that the reduction of GBSS transcript expression under HT was generally accompanied by the decreased expressions of SSSIIa, SSSIIIa and SBEIIb. Consequently, high temperature-ripened grains in 9311 eha showed high proportion of intermediate and long B chains and somewhat lower level of short A chain compared to the wildtype. The temperature-dependent alteration of amylose content was not only attributed to the reduced expression of GBSS, but also associated with the complimentary effect of SSSIIa and SBEIIb.

Genotypic-dependent alteration in transcriptional expression of various CAT isoenzyme genes in esl mutant rice and its relation to H2O2-induced leaf senescence

To clarify the intricate relationship of H2O2-induced leaf senescence with distinct CAT gene expression, the genotypic-dependent alteration in the temporal patterns of H2O2 content, malondialdehyde (MDA) accumulation, relative conductivity, and the transcript expression of three CAT isoform genes during leaf senescence was investigated using two rice genotypes, namely, the early senescence leaf (esl) mutant and its wild type. Detached leaf segments were used to examine the effects of different exogenous H2O2 and ABA treatments on the expression of three CAT genes and other senescence-related physiological parameters. The results showed that the esl mutant had higher H2O2 level than its wild type at the initial phase of leaf senescence, which was followed by a sharp increase in MDA accumulation. Then, membrane integrity was severely destroyed and chlorophyll content decreased rapidly, which accelerated the senescence symptoms and significantly decreased the seed-setting rate in the esl mutant. The association of expression of the three CAT genes with rice leaf senescence lesions induced by H2O2 was isoform dependent, and the CATA and CATB expression exhibited remarkable genotype-dependent variation in the amount of transcript and their temporal pattern during leaf senescence. CATA and CATB also showed more sensitive response to exogenous H2O2 treatment compared with CATC, which suggests that CATA and CATB genes have a more important role than CATC in H2O2 inactivation of rice leaf senescence. The contribution of CATA expression to H2O2 scavenging in senescing rice leaves was most prominent at the initial stage of leaf senescence, with higher levels observed at the onset of leaf senescence, whereas CATB expression in esl flag leaf peaked later compared with CATA and was also responsible for the execution and acceleration of H2O2 inactivation particularly after the initiation of leaf senescence. CATB expression partially overlapped with CATA expression during H2O2 scavenging. For the CATC expression in rice leaves, its temporal pattern was poorly associated with H2O2-induced leaf senescence, but CATC was expressed preferentially in rice leaves and sheath and was involved in organ/tissue specificity. Exogenous ABA treatment can cause senescence-related increase in MDA accumulation and relative conductivity in the detached leaf segments, concomitant with a remarkable increase in H2O2 level. However, the expression of the three CAT isoforms under exogenous ABA treatment differed from those induced directly by H2O2 treatment, indicating the different ways for scavenging H2O2 accumulation during leaf senescence despite the increase in H2O2 level in senescing leaves induced by ABA.

Relationship of ROS accumulation and superoxide dismutase isozymes in developing anther with floret fertility of rice under heat stress

High temperature (HT) at meiosis stage is one of most important environment constraint affecting spikelet fertility and rice yield. In this paper, the effects of HT exposure at meiosis stage on the ROS (reactive oxygen species) accumulation, various superoxide dismutase (SOD, EC1.15.1.11) isozymes in developing anther, and its relationship with HT-induced decline in pollen viability and floret fertility were investigated by using four rice cultivars differing in heat tolerance under well-controlled climatic condition. Results showed that HT exposure significantly increased ROS level and malondialdehyde (MDA) content in rice anther, and this occurrence was strongly responsible for the HT-induced decline in pollen viability and harmful effect of HT adversity on floret fertility. However, the increased extent of ROS concentration in rice anther under HT exposure was greatly variable, depending on both the intensity and duration of HT exposure and different rice cultivars used. The SOD and CAT activities of HT-sensitive cultivars decreased more profoundly than those of HT-tolerant cultivars under the same HT regimes. Among various types of SOD enzymes, Cu/Zn-SODa expressed highly in rice anther and responded sensitively to HT exposure, while Cu/Zn-SODb expressed weakly in rice anther and preferentially in rice leaves. HT exposure suppressed the expression of Cu/Zn-SODa in developing anther, which was closely associated with the down-regulated transcripts of cCu/Zn-SOD1 gene. Hence, Cu/Zn-SODa may play a central role in the regulation of total SOD activity and ROS detoxification in rice anther as affected by HT exposure at meiosis stage.

Positional variation in grain mineral nutrients within a rice panicle and its relation to phytic acid concentration

Six japonica rice genotypes, differing in panicle type, grain density, and phytic acid (PA) content, were applied to investigate the effect of grain position on the concentrations of major mineral nutrients and its relation to PA content and grain weight within a panicle. Grain position significantly affected the concentrations of the studied minerals in both the vertical and horizontal axes of a rice panicle. Heavy-weight grains, located on primary rachis and top rachis, generally had higher mineral concentrations, but were lower in PA concentration and molar ratios of PA/Zn, compared with the small-weight grains located on secondary rachis and bottom rachis, regardless of rice genotypes. However, on the basis of six rice genotypes, no significant correlations were found among mineral elements, PA, and grain weight. These results suggested that some desired minerals, like Zn and Fe, and their bioavailability, can be enhanced simultaneously by the modification of panicle patterns, and it will be helpful in the selection of rice genotypes with low PA and high mineral nutrients for further breeding strategy without sacrificing their high yields.概要研究目的阐明水稻穗内不同粒位间的主要矿质营养元素和植酸含量差异、 粒位分布特点及其与品种穗型间的联系。创新要点将水稻品种的穗型变化与稻米营养品质结合起来, 从水稻穗粒结构角度, 对同一稻穗内不同籽粒间的主要矿质营养元素与植酸含量差异、 粒位分布特点及其与水稻品种穗型间的相互关系进行了较系统的探讨分析。研究方法以典型的直立穗型和弯穗型粳稻品种为材料, 通过对两类水稻品种在相同栽培条件下籽粒矿质营养元素和植酸含量的测定分析, 并依据水稻籽粒在稻穗上的着生部位, 将同一稻穗内的不同籽粒划分为六个粒位, 比较分析了两类品种同一稻穗内不同部位间矿质营养元素和植酸含量的差异变化及其粒位分布特点。重要结论水稻穗型虽然与品种间的籽粒矿质营养元素和植酸含量高低没有直接关系, 但对其穗内不同籽粒间的主要矿质营养元素和植酸含量存在着较大影响; 与稻穗中下部的弱势粒相比, 同一稻穗内着生在稻穗上中部的强势粒通常具有相对较高的锌、 铁矿质元素含量, 而籽粒植酸含量和植酸/锌(铁)摩尔比则有所降低, 稻米营养品质也相对较好; 不同矿质营养元素相比, 粒位效应对铁矿质营养的影响作用要略大于对钙和锌营养元素含量。