Protein Disulfide Isomerase-Like Protein 1-1 Controls Endosperm Development through Regulation of the Amount and Composition of Seed Proteins in Rice

Protein disulfide isomerase (PDI) is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER). A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1) during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species) scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.     

Seeds of tomato (Lycopersicon esculentum Mill.) which develop in a fully hydrated environment in the fruit switch from a developmental to a germinative mode without a requirement for desiccation

Seed water content is high during early development of tomato seeds (10–30 d after pollination (DAP)), declines at 35 DAP, then increases slightly during fruit ripening (following 50 DAP). The seed does not undergo maturation drying. Protein content during seed development peaks at 35 DAP in the embryo, while in the endosperm it exhibits a triphasic accumulation pattern. Peaks in endosperm protein deposition correspond to changes in endosperm morphology (i.e. formation of the hard endosperm) and are largely the consequence of increases in storage proteins. Storage-protein deposition commences at 20 DAP in the embryo and endosperm; both tissues accumulate identical proteins. Embryo maturation is complete by 40 DAP, when maximum embryo protein content, size and seed dry weight are attained. Seeds are tolerant of premature drying (fast and slow drying) from 40 DAP.Thirty-and 35-DAP seeds when removed from the fruit tissue and imbibed on water, complete germination by 120 h after isolation. Only seeds which have developed to 35 DAP produce viable seedlings. The inability of isolated 30-DAP seed to form viable seedlings appears to be related to a lack of stored nutrients, since the germinability of excised embryos (20 DAP and onwards) placed on Murashige and Skoog (1962, Physiol. Plant. 15, 473–497) medium is high. The switch from a developmental to germinative mode in the excised 30- and 35-DAP imbibed seeds is reflected in the pattern of in-vivo protein synthesis. Developmental and germinative proteins are present in the embryo and endosperm of the 30- and 35-DAP seeds 12 h after their isolation from the fruit. The mature seed (60 DAP) exhibits germinative protein synthesis from the earliest time of imbibition. The fruit environment prevents precocious germination of developing seeds, since the switch from development to germination requires only their removal from the fruit tissue.Abbreviations DAP days after pollination - kDa kilodaltons - SP1-4 storage proteins 1–4 - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - HASI hours after seed isolation - MS medium Murashige and Skoog (1962) medium This work is supported by National Science and Engineering Research Council of Canada grant A2210 to J.D.B.     

RACK1 regulates specific functions of Gbetagamma

We showed previously that Gbetagamma interacts with Receptor for Activated C Kinase 1 (RACK1), a protein that not only binds activated protein kinase C (PKC) but also serves as an adaptor/scaffold for many signaling pathways. Here we report that RACK1 does not interact with Galpha subunits or heterotrimeric G proteins but binds free Gbetagamma subunits released from activated heterotrimeric G proteins following the activation of their cognate receptors in vivo. The association with Gbetagamma promotes the translocation of RACK1 from the cytosol to the membrane. Moreover, binding of RACK1 to Gbetagamma results in inhibition of Gbetagamma-mediated activation of phospholipase C beta2 and adenylyl cyclase II. However, RACK1 has no effect on other functions of Gbetagamma, such as activation of the mitogen-activated protein kinase signaling pathway or chemotaxis of HEK293 cells via the chemokine receptor CXCR2. Similarly, RACK1 does not affect signal transduction through the Galpha subunits of G(i), G(s), or G(q). Collectively, these findings suggest a role of RACK1 in regulating specific functions of Gbetagamma.     

The betagamma subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins

A yeast two-hybrid approach was used to discern possible new effectors for the betagamma subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gbeta, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gbetagamma using an anti-Gbeta antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gbeta(1)gamma(1) but also unexpectedly with the transducin heterotrimer Galpha(t)beta(1)gamma(1). Galpha(t) alone does not interact, but it must contribute to the interaction because the apparent EC(50) value of RACK1 for Galpha(t)beta(1)gamma(1) is 3-fold greater than that for Gbeta(1)gamma(1) (0.1 versus 0.3 microm). RACK1 is a scaffold that interacts with several proteins, among which are activated betaIIPKC and dynamin-1 (1). betaIIPKC and dynamin-1 compete with Gbeta(1)gamma(1) and Galpha(t)beta(1)gamma(1) for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gbetagamma interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.     

Polarity exchange at the interface of regulators of G protein signaling with G protein alpha-subunits

RGS proteins are GTPase-activating proteins (GAPs) for G protein alpha-subunits. This GAP activity is mediated by the interaction of conserved residues on regulator of G protein signaling (RGS) proteins and Galpha-subunits. We mutated the important contact sites Glu-89, Asn-90, and Asn-130 in RGS16 to lysine, aspartate, and alanine, respectively. The interaction of RGS16 and its mutants with Galpha(t) and Galpha(i1) was studied. The GAP activities of RGS16N90D and RGS16N130A were strongly attenuated. RGS16E89K increased GTP hydrolysis of Galpha(i1) by a similar extent, but with an about 100-fold reduced affinity compared with non-mutated RGS16. As Glu-89 in RGS16 is interacting with Lys-210 in Galpha(i1), this lysine was changed to glutamate for compensation. Galpha(i1)K210E was insensitive to RGS16 but interacted with RGS16E89K. In rat uterine smooth muscle cells, wild type RGS16 abolished G(i)-mediated alpha(2)-adrenoreceptor signaling, whereas RGS16E89K was without effect. Both Galpha(i1) and Galpha(i1)K210E mimicked the effect of alpha(2)-adrenoreceptor stimulation. Galpha(i1)K210E was sensitive to RGS16E89K and 10-fold more potent than Galpha(i1). Analogous mutants of Galpha(q) (Galpha(q)K215E) and RGS4 (RGS4E87K) were created and studied in COS-7 cells. The activity of wild type Galpha(q) was counteracted by wild type RGS4 but not by RGS4E87K. The activity of Galpha(q)K215E was inhibited by RGS4E87K, whereas non-mutated RGS4 was ineffective. We conclude that mutation of a conserved lysine residue to glutamate in Galpha(i) and Galpha(q) family members renders these proteins insensitive to wild type RGS proteins. Nevertheless, they are sensitive to glutamate to lysine mutants of RGS proteins. Such mutant pairs will be helpful tools in analyzing Galpha-RGS specificities in living cells.     

Proteomic study identifies proteins involved in brassinosteroid regulation of rice growth

Brassinosteroids (BRs) are essential hormones for growth and development of plant. In rice, BRs regulate multiple developmental processes and affect many important traits such as height, leaf angle, fertility and seed filling. We identified brassinosteroid-regulated proteins in rice using proteomic approaches and performed functional analysis of some BR-regulated proteins by overexpression experiments. Using two-dimensional difference gel electrophoresis (2-D DIGE) followed by protein identification by mass spectrometry, we compared proteomic differences in the shoots and roots of the BR-insensitive mutant d61-4 and BR-deficient mutant brd1-3. We identified a large number of proteins differentially expressed in the mutants compared with wild type control. These include a glycine-rich RNA-binding protein (OsGRP1) and a DREPP2 protein, which showed reduced levels in the BR mutants. Overexpression of these two proteins partially suppressed the dwarf phenotype of the Arabidopsis BR-insensitive mutant bri1-5. In contrast to the reduced protein level, the RNA level of OsGRP1 was not significantly affected in the BR mutants or by BR treatment, suggesting BR regulation of OsGRP1 at the posttranslational level. This study identifies many BR-regulated proteins and demonstrates that OsGRP1 functions downstream in the BR signal transduction pathway to promote cell expansion.     

Alterations by a defect in a rice G protein α subunit in probenazole and pathogen-induced responses

The rice dwarf1 (d1) mutant, which is deficient in an α subunit (Gα) of heterotrimeric G protein, was used to obtain specific evidence on the functions of Gα protein in defence signalling in rice. Using proteome analysis, a probenazole‐inducible protein (PBZ1) was detected in the cytosolic fraction of leaf blade of the wild type, but not the d1 mutant. After treatment with probenazol, PBZ1 reached maximal levels at 72 h in the wild type but 96 h in the d1 mutant. The induction of PBZ1 by probenazole treatment was inhibited by protein kinase inhibitors. A 48‐kDa putative mitogen‐activated protein kinase (MAPK) and a 55‐kDa putative Ca²⁺‐dependent protein kinase (CDPK) showed lower activities in the cytosolic fraction of the d1 mutant than that of the wild type. The activities of these protein kinases were enhanced at 24 h in the wild type and 48 h in the d1 mutant after probenazole treatment. Although the d1 mutant responded to the rice blast fungus similarly to the wild type, the d1 mutant developed rice blight symptoms earlier than the wild type when infected with Xoo. In addition, the blight symptoms were more severe on the mutant than on the wild type, and wilting was frequently observed in the d1 mutant. Furthermore, induction by the bacterial infection of the 48‐kDa putative MAPK and PBZ1 was delayed by 2 and 4 d, respectively, in the d1 mutant compared with the wild type. These results indicate that the Gα protein plays a role in the induction of PBZ1 and protein kinases by probenazole and Xoo, and suggest that the 48‐kDa putative MAPK may be involved in a signalling pathway for resistance to bacterial infection.     

Rice alcohol dehydrogenase 1 promotes survival and has a major impact on carbohydrate metabolism in the embryo and endosperm when seeds are germinated in partially oxygenated water

Background and Aims

Rice (Oryza sativa) has the rare ability to germinate and elongate a coleoptile under oxygen-deficient conditions, which include both hypoxia and anoxia. It has previously been shown that ALCOHOL DEHYDROGENASE 1 (ADH1) is required for cell division and cell elongation in the coleoptile of submerged rice seedlings by means of studies using a rice ADH1-deficient mutant, reduced adh activity (rad). The aim of this study was to understand how low ADH1 in rice affects carbohydrate metabolism in the embryo and endosperm, and lactate and alanine synthesis in the embryo during germination and subsequent coleoptile growth in submerged seedlings.

Methods

Wild-type and rad mutant rice seeds were germinated and grown under complete submergence. At 1, 3, 5 and 7 d after imbibition, the embryo and endosperm were separated and several of their metabolites were measured and compared.

Key results

In the rad embryo, the rate of ethanol fermentation was halved, while lactate and alanine concentrations were 2·4- and 5·7- fold higher in the mutant than in the wild type. Glucose and fructose concentrations in the embryos increased with time in the wild type, but not in the rad mutant. The rad mutant endosperm had lower amounts of the α-amylases RAMY1A and RAMY3D, resulting in less starch degradation and lower glucose concentrations.

Conclusions

These results suggest that ADH1 is essential for sugar metabolism via glycolysis to ethanol fermentation in both the embryo and endosperm. In the endosperm, energy is presumably needed for synthesis of the amylases and for sucrose synthesis in the endosperm, as well as for sugar transport to the embryo.     

On the Composition, Deposition and Mobilization of Proteins in the Cotyledons of Castor Bean (Ricinus communis L. cv. Hale) Seeds: Their Role as Storage Proteins

Proteins in the soluble and insoluble fractions, extracted frommature castor bean cv. Hale seed cotyledons, differ quantitativelyand qualitatively from their counterparts extracted from theendosperm. The soluble fraction contains no glycoproteins, andthe lectins RCA₁ and ricin D are absent. While the insolubleproteins are electrophoretically and immunologically similarto those in the endosperm, they do not form the 100 kD subunitdimers which characterize some of the endosperm insoluble crystalloidproteins. Rapid rates of deposition of all of the soluble andinsoluble proteins present in the mature seed cotyledons commences30–35 d after pollination (DAP) and continues until 45DAP. These proteins are mobilized rapidly beginning 1–2d after seed imbibition and this coincides with an increasein specific activity, in the cotyledons, of two aminopeptidasesand a carboxypeptidase. The soluble and insoluble proteins inthe cotyledons of the mature seed probably function as storageproteins and support the growth of the germinated seed priorto the mobilization of the major protein storage reserves ofthe endosperm. Key words: Ricinus communis, Castor bean, Hale cultivar, Cotyledon, Storage protein, Seed development, Seed germination     

Temporal and spatial expression pattern of the OSVP1 and OSEM genes during seed development in rice

The spatial and temporal expression patterns of the rice VP1 (OSVP1) gene, as well as the OSEM gene which it controls, were studied during seed development by in situ hybridization and immuno-localization techniques. The expression of OSVP1 could be detected in embryos as early as 2-3 d after pollination (DAP) and thereafter became preferentially localized to shoot, radicle and vascular tissues during the embryo development at both the mRNA and protein levels. In the aleurone layers, OSVP1 mRNA and protein were detected after 6 DAP. OSEM mRNA was detectable after 6 DAP in the embryo and aleurone tissue. The spatial distribution within the embryo of OSEM mRNA and OSVP1 mRNA/protein was very similar after 6 DAP. Transgenic rice carrying a beta-glucuronidase (GUS) gene transcribed from a chimeric promoter consisting of the CaMV 35S minimal promoter (-46) and the 55-bp promoter fragment of OSEM, minimally required for ABA and VP1 regulation, also exhibited a spatial pattern of GUS expression similar to that of OSEM and OSVP1. These results suggest that (OS)VP1 is a major determinant not only of the seed specificity but also of the spatial pattern of OSEM expression in the developing seed.     

Morphological analysis of seed shape in Arabidopsis thaliana reveals altered polarity in mutants of the ethylene signaling pathway

The shape of Arabidopsis thaliana dry seed is described here as a prolate spheroid. The accuracy of this approximation is discussed. Considering its limitations, it allows a geometric approximation to the analysis of changes occurring in seed shape during imbibition prior to seed germination as well as the differences in shape between genotypes and their changes during imbibition. The triple mutant ein2-1, ers1-2, etr1-7 presents notable alterations in seed shape. In addition, seeds of this and other mutants in the ethylene signaling pathway (ctr1-1, eto1-1, etr1-1, ein2-1) show different response to imbibition than the wild type. Imbibed seeds of the wild type increase their asymmetry compared with the dry seeds. This is detected by the relative changes in the curvature values in both poles. Thus, during imbibition of the wild-type seeds, the reduction in curvature values observed in the basal pole gives them an ovoid shape. In contrast, in the seeds of the ethylene mutants, reduction in curvature values occurs in both basal and apical poles, and its shape remains as a prolate spheroid. Our data indicate that the ethylene signaling pathway is involved, in general, in the complex regulation of seed shape and, in particular, in the establishment of polarity in seeds, controlling curvature values in the seed poles.     

Spatio-temporal changes in germination and radical elongation of barley seeds tracked by proteome analysis of dissected embryo, aleurone layer, and endosperm tissues

Germination of barley is accompanied by changes in water-soluble seed proteins. 2-DE was used to describe spatio-temporal proteome differences in dissected seed tissues associated with germination and the subsequent radicle elongation. Protein identification by MS enabled assignment of proteins and functions to the seed embryo, aleurone, and endosperm. Abundance in 2-DE patterns was monitored for 48 different proteins appearing in 79 gel spots at 8 time-points up to 72 h post imbibition (PI). In embryo, a beta-type proteasome subunit and a heat shock protein 70 fragment were among the earliest proteins to appear (at 4 h PI). Other early changes were observed that affected spots containing desiccation stress-associated late embryogenesis abundant and abscisic acid (ABA)-induced proteins. From 12 h PI proteins characteristic for desiccation stress disappeared rapidly, as did a putative embryonic protein and an ABA-induced protein, suggesting that these proteins are also involved in desiccation stress. Several redox-related proteins differed in spatio-temporal patterns at the end of germination and onset of radicle elongation. Notably, ascorbate peroxidase that was observed only in the embryo, increased in abundance at 36 h PI. The surprisingly early changes seen in the protein profiles already 4 h after imbibition indicate that germination is programmed during seed maturation.     

赤霉素与脱落酸对番茄种子萌发中细胞周期的调控

利用细胞流检仪检测番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．） ＧＡ－缺陷型、ＡＢＡ－缺陷型和相应的正常品种（野生型）成熟种子胚根尖细胞倍性水平时发现：ＧＡ－缺陷型和野生型种子绝大多数细胞ＤＮＡ 水平为２Ｃ,而ＡＢＡ－缺陷型种子则含有较多的４Ｃ细胞。在标准发芽条件下,ＡＢＡ－缺陷型和野生型种子浸种１ ｄ 后胚根尖细胞ＤＮＡ 开始复制,随后胚根突破种皮而发芽。然而ＧＡ－缺陷型种子除非加入外源ＧＡ,否则既不发生细胞ＤＮＡ 复制,也不发芽。这说明内源ＧＡ 是启动番茄种子胚根尖细胞ＤＮＡ 复制的关键因素,同时也说明番茄根尖细胞ＤＮＡ 复制是种子发芽的必要条件。实验证明：ＡＢＡ 不抑制细胞ＤＮＡ 合成,但阻止Ｇ２ 细胞进入到Ｍ 期。外源ＡＢＡ处理野生型种子与渗控处理结果相似,可以大幅度提高胚根尖４Ｃ／２Ｃ细胞的比例,但抑制种子的最终发芽     

GA and ABA Regulation on the Cell Cycle in Germination of Tomato Seeds

Flow cytometric determination of ploidy levels in embryos of GA-deficient, ABA-deficient mutant and isogenic wild type tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds revealed that, large amount of 2C DNA signals existed both in wild type and GA-deficient mutant seeds, showing that most cells had arrested in the cell cycle at presynthesis Gl, whereas a relative amount of 4C proportion which is a sign of seed germination was found in ABA-deficient mutant seeds, indicating that endogenous ABA play a role in regulating the switch from development to germination in seeds. DNA replication was stimulated 1 d after the seed was imbibed in water and a visible germination occurred subsequently either in wild type GA-deficient mutant seeds. But it was not the case for ABA-deficient mutant seeds unless an exogenous GA was supplemented. This demonstrated that DNA replication in embryo root tips cells was subjected to be a compulsory factor for seed germination, whereas endogenous GA triggered DNA synthesis. It was evident that exogenous ABA could inhibit seed germination not by suppressing DNA synthesis but by bloking the route leading to mitosis since a great amount of 4C proportion was found in the germinating wild type and GA-deficient mutant seeds in the ABA solution when visible ger mination did not occur. Finally a simple mode of hormonal regulation on cell cycle in high plants was hypothesized.     

Gib2, a novel Gbeta-like/RACK1 homolog, functions as a Gbeta subunit in cAMP signaling and is essential in Cryptococcus neoformans

Canonical G proteins are heterotrimeric, consisting of alpha, beta, and gamma subunits. Despite multiple Galpha subunits functioning in fungi, only a single Gbeta subunit per species has been identified, suggesting that non-conventional G protein signaling exists in this diverse group of eukaryotic organisms. Using the Galpha subunit Gpa1 that functions in cAMP signaling as bait in a two-hybrid screen, we have identified a novel Gbeta-like/RACK1 protein homolog, Gib2, from the human pathogenic fungus Cryptococcus neoformans. Gib2 contains a seven WD-40 repeat motif and is predicted to form a seven-bladed beta propeller structure characteristic of beta transducins. Gib2 is also shown to interact, respectively, with two Ggamma subunit homologs, Gpg1 and Gpg2, similar to the conventional Gbeta subunit Gpb1. In contrast to Gpb1 whose overexpression promotes mating response, overproduction of Gib2 suppresses defects of gpa1 mutation in both melanization and capsule formation, the phenotypes regulated by cAMP signaling and associated with virulence. Furthermore, depletion of Gib2 by antisense suppression results in a severe growth defect, suggesting that Gib2 is essential. Finally, Gib2 is shown to also physically interact with a downstream target of Gpa1-cAMP signaling, Smg1, and the protein kinase C homolog Pkc1, indicating that Gib2 is also a multifunctional RACK1-like protein.     

The SMALL AND ROUND SEED1 (SRS1/DEP2) gene is involved in the regulation of seed size in rice

The causal gene of a novel small and round seed mutant 1 (srs1) was identified in rice by map-based cloning and named SMALL AND ROUND SEED 1 (SRS1). The SRS1 gene is identical to the previously identified DENSE AND ERECT PANICLE 2 (DEP2). The SRS1/DEP2 gene encodes a novel protein of 1365 amino acids residues without known functional domains. In the longitudinal direction of the lemma, both cell length and cell number are reduced in srs1-1 compared to the wild type, whereas in the lateral cross section of the lemma, cell length in srs1-1 is greater than that in the wild type, but the cell number in srs1-1 is the same as that in wild type. These results suggest that the small and round seed phenotype of srs1-1 is due to the reduction in both cell length and cell number in the longitudinal direction, and the elongation of the cells in the lateral direction of the lemma. The SRS1 mRNA and proteins are abundant in wild type rice specifically in young organs, namely young leaves, internodes and panicles. Interestingly, the tissues expressing SRS1 are closely related to the tissues that exhibit abnormalities in the srs1 mutants.     

A Comparative Study on the Role of Cytokinins in Caryopsis Development in the Maize miniature1 Seed Mutant and Its Wild Type

We report here on a comparative developmental profile of plant hormone cytokinins in relation to cell size, cell number and endoreduplication in developing maize caryopsis of a cell wall invertase-deficient miniature1 ( mn1 ) seed mutant and its wild type, Mn1 , genotype. Both genotypes showed extremely high levels of total cytokinins during the very early stages of development, followed by a marked and genotype specific reduction. While the decrease of cytokinins in Mn1 was associated with their deactivation by 9-glucosylation, the absolute and the relative part of active cytokinin forms was higher in the mutant. During the exponential growth phase of endosperm between 6 d after pollination and 9 d after pollination, the mean cell doubling time, the absolute growth rate and the level of endoreduplication were similar in the two genotypes. However, the entire duration of growth was longer in Mn1 compared with mn1 , resulting in a significantly higher cell number in the Mn1 endosperm. These data correlate with the previously reported peak levels of the Mn1 -encoded cell wall invertase-2 (INCW2) at 12 d after pollination in the Mn1 endosperm. A model showing possible crosstalk among cytokinins, cell cycle and cell wall invertase as causal to increased cell number and sink strength of the Mn1 developing endosperm is discussed.