Chaperone-like properties of tobacco plastid thioredoxins f and m

Thioredoxins (Trxs) are ubiquitous disulphide reductases that play important roles in the redox regulation of many cellular processes. However, some redox-independent functions, such as chaperone activity, have also been attributed to Trxs in recent years. The focus of our study is on the putative chaperone function of the well-described plastid Trxs f and m. To that end, the cDNA of both Trxs, designated as NtTrxf and NtTrxm, was isolated from Nicotiana tabacum plants. It was found that bacterially expressed tobacco Trx f and Trx m, in addition to their disulphide reductase activity, possessed chaperone-like properties. In vitro, Trx f and Trx m could both facilitate the reactivation of the cysteine-free form of chemically denatured glucose-6 phosphate dehydrogenase (foldase chaperone activity) and prevent heat-induced malate dehydrogenase aggregation (holdase chaperone activity). Our results led us to infer that the disulphide reductase and foldase chaperone functions prevail when the proteins occur as monomers and the well-conserved non-active cysteine present in Trx f is critical for both functions. By contrast, the holdase chaperone activity of both Trxs depended on their oligomeric status: the proteins were functional only when they were associated with high molecular mass protein complexes. Because the oligomeric status of both Trxs was induced by salt and temperature, our data suggest that plastid Trxs could operate as molecular holdase chaperones upon oxidative stress, acting as a type of small stress protein

The Mitochondrial Cycle of Arabidopsis Shoot Apical Meristem and Leaf Primordium Meristematic Cells Is Defined by a Perinuclear Tentaculate/Cage-Like Mitochondrion1[W][OA]

Plant cells exhibit a high rate of mitochondrial DNA (mtDNA) recombination. This implies that before cytokinesis, the different mitochondrial compartments must fuse to allow for mtDNA intermixing. When and how the conditions for mtDNA intermixing are established are largely unknown. We have investigated the cell cycle-dependent changes in mitochondrial architecture in different Arabidopsis (Arabidopsis thaliana) cell types using confocal microscopy, conventional, and three-dimensional electron microscopy techniques. Whereas mitochondria of cells from most plant organs are always small and dispersed, shoot apical and leaf primordial meristematic cells contain small, discrete mitochondria in the cell periphery and one large, mitochondrial mass in the perinuclear region. Serial thin-section reconstructions of high-pressure-frozen shoot apical meristem cells demonstrate that during G1 through S phase, the large, central mitochondrion has a tentaculate morphology and wraps around one nuclear pole. In G2, both types of mitochondria double their volume, and the large mitochondrion extends around the nucleus to establish a second sheet-like domain at the opposite nuclear pole. During mitosis, approximately 60% of the smaller mitochondria fuse with the large mitochondrion, whose volume increases to 80% of the total mitochondrial volume, and reorganizes into a cage-like structure encompassing first the mitotic spindle and then the entire cytokinetic apparatus. During cytokinesis, the cage-like mitochondrion divides into two independent tentacular mitochondria from which new, small mitochondria arise by fission. These cell cycle-dependent changes in mitochondrial architecture explain how these meristematic cells can achieve a high rate of mtDNA recombination and ensure the even partitioning of mitochondria between daughter cells

Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures

In this work we address two aspects of eggplant flower biology potentially involved on the efficiency of anther culture: the selection of the best floral stage to extract anthers for culture, and the effect of heterostyly in the identification of suitable buds and anthers. For 12 different accessions, we determined morphological criteria (length ranges) to identify buds and anthers enriched in vacuolate microspores and young bicellular pollen, the stages most responsive to embryogenesis induction. While these microspore/pollen stages were the most responsive when isolated and cultured in liquid medium, we observed that culture of anthers containing these stages is not the best choice. Instead, the highest response was found for younger anthers, containing mostly young and mid microspores. We analyzed eggplant anther walls and found that their particular thickness may be behind this apparent discrepancy, since they may delay the diffusion of inducing factors to the anther locule, reducing their effect over inducible microspores. Thus, the culture of younger anthers would allow for younger microspores to grow up to the inducible stages while factors are entering the locule. We also analyzed the embryogenic response of short and long-styled buds present in Cristal, a heterostylic cultivar. Our results demonstrated that each floral morph produced buds and anthers of different lengths, but equally useful for anther culture, since similar amounts of embryos were produced. The practical application of these results may improve the efficiency of anther culture not only in these cultivars, but in others also presenting thick anther walls and heterostyly. © 2011 Springer Science+Business Media B.V.We want to acknowledge Drs. Begona Renau and Antonio Serrano, and Mrs Nuria Palacios for their excellent technical work, as well as the staff of the COMAV greenhouses for their valuable help. We also thank the anonymous reviewers for their excellent criticisms and the ideas they contributed. This work was supported by grant AGL2010-17895 from Spanish MICINN to JMSS.Salas Aragon, P.; Rivas Sendra, A.; Prohens Tomás, J.; Seguí-Simarro, JM. (2012). Influence of the stage for anther excision and heterostyly in embryogenesis induction from eggplant anther cultures. Euphytica. 184(2):235-250. https://doi.org/10.1007/s10681-011-0569-9S235250184

Electron Tomographic Analysis of Somatic Cell Plate Formation in Meristematic Cells of Arabidopsis Preserved by High-Pressure Freezing

We have investigated the process of somatic-type cytokinesis in Arabidopsis (Arabidopsis thaliana) meristem cells with a three-dimensional resolution of ∼7 nm by electron tomography of high-pressure frozen/freeze-substituted samples. Our data demonstrate that this process can be divided into four phases: phragmoplast initials, solid phragmoplast, transitional phragmoplast, and ring-shaped phragmoplast. Phragmoplast initials arise from clusters of polar microtubules (MTs) during late anaphase. At their equatorial planes, cell plate assembly sites are formed, consisting of a filamentous ribosome-excluding cell plate assembly matrix (CPAM) and Golgi-derived vesicles. The CPAM, which is found only around growing cell plate regions, is suggested to be responsible for regulating cell plate growth. Virtually all phragmoplast MTs terminate inside the CPAM. This association directs vesicles to the CPAM and thereby to the growing cell plate. Cell plate formation within the CPAM appears to be initiated by the tethering of vesicles by exocyst-like complexes. After vesicle fusion, hourglass-shaped vesicle intermediates are stretched to dumbbells by a mechanism that appears to involve the expansion of dynamin-like springs. This stretching process reduces vesicle volume by ∼50%. At the same time, the lateral expansion of the phragmoplast initials and their CPAMs gives rise to the solid phragmoplast. Later arriving vesicles begin to fuse to the bulbous ends of the dumbbells, giving rise to the tubulo-vesicular membrane network (TVN). During the transitional phragmoplast stage, the CPAM and MTs disassemble and then reform in a peripheral ring phragmoplast configuration. This creates the centrifugally expanding peripheral cell plate growth zone, which leads to cell plate fusion with the cell wall. Simultaneously, the central TVN begins to mature into a tubular network, and ultimately into a planar fenestrated sheet (PFS), through the removal of membrane via clathrin-coated vesicles and by callose synthesis. Small secondary CPAMs with attached MTs arise de novo over remaining large fenestrae to focus local growth to these regions. When all of the fenestrae are closed, the new cell wall is complete. Few endoplasmic reticulum (ER) membranes are seen associated with the phragmoplast initials and with the TVN cell plate that is formed within the solid phragmoplast. ER progressively accumulates thereafter, reaching a maximum during the late PFS stage, when most cell plate growth is completed

Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.)

Production of doubled haploids (DHs) through androgenesis induction is an important biotechnological tool for plant breeding. In some species, DHs are efficiently obtained through embryogenesis from isolated microspore cultures. In eggplant, however, this process is still at its infancy, despite the economic relevance of this important agricultural crop. To date, only two studies have focused previously on this process, suggesting that in eggplant microspore cultures, the only morphogenic response is callus formation. Given the notable lack of studies on eggplant microspore cultures, in this work we explored this process with different experimental approaches. We studied the response of different cultivars and characterized the development of microspores induced to divide and proliferate. We demonstrated that microspore-derived embryos (MDEs) can be produced in eggplant; however, MDEs stopped at the globular stage, to turn into euploid and principally mixoploid calli. From these calli, 60 % of DH plants could be regenerated. In order to promote microspore induction we evaluated the effect of polyethylene glycol (PEG) and mannitol. PEG, but not mannitol, significantly increased induction of microspore embryogenesis. We also tested the ability of eight different media compositions to promote efficient plant regeneration from calli. In order to test it in a genotype-independent manner, we previously developed a method to generate clonal callus populations derived from single microspore-derived calli. Together, the results presented hereby constitute an efficient way to produce eggplant DHs through microspore culture. In addition, they contribute significant insights into the knowledge of the particularities of androgenesis induction in this species. © 2012 Springer Science+Business Media B.V.We acknowledge Mrs. Nuria Palacios for her excellent technical work, as well as the staff of the COMAV greenhouses for their valuable help. Thanks are also due to Dr. Santiago Vilanova for providing us with the SSRs used in this work. This work was supported by grant from Spanish MICINN AGL2010-17895 to JMSS.Corral Martínez, P.; Seguí-Simarro, JM. (2012). Efficient production of callus-derived doubled haploids through isolated microspore culture in eggplant (Solanum melongena L.). Euphytica. 187(1):47-61. https://doi.org/10.1007/s10681-012-0715-zS4761187

The use of corms produced under storage at low temperatures as a sourceof explants for the in vitro propagation of saffron reduces contaminationlevels and increases multiplication rates

Saffron is a triploid sterile crop with a low vegetative propagation rate, which prevents the widespread use of selected genotypes. Despite efficient in vitro propagation via direct organogenesis being very interesting, high contamination levels and low propagation rates preclude the commercial use of this technique. In order to obtain a source of explants for the in vitro multiplication of saffron that allows low contamination levels and efficient propagation, daughter corms were produced by storing non-planted mother corms at low temperatures (1-3 degrees C) for 9 months. This method avoids field cultivation and yields more corms than field production (8 vs. 3 corms per mother corm). Corms produced under cold storage conditions were in vitro sprouted on media with a high level of cytokinins (5 mg l(-1) 6-benzylaminopurine or 1 mg l(-1) thidiazuron) and 0.5 mg l(-1) naphthalene acetic acid, and sprouted buds were cultivated on the same media. After 8 weeks of culture, multiple shoot primordia emerged from the base of the sprouted buds with no callus formation, and up to 400 shoot primordia were produced from one initial mother corm. These shoot primordia can be elongated and 90% produce corms. The high multiplication rate, lack of contamination, and the fact that multiplication occurs through direct organogenesis make the method suitable for the propagation of selected genotypes. Otherwise shoot clusters can be maintained for 6-7 months. However for longer maintenance periods, shoot proliferation capacity by direct organogenesis diminishes, and the emergence of somaclonal variation cannot be ruled out.Renau Morata, B.; Moya, L.; González Nebauer, S.; Seguí-Simarro, JM.; Parra Vega, V.; Gómez Jiménez, MD.; Molina Romero, RV. (2013). The use of corms produced under storage at low temperatures as a sourceof explants for the in vitro propagation of saffron reduces contaminationlevels and increases multiplication rates. Industrial Crops and Products. 46:97-104. doi:10.1016/j.indcrop.2013.01.013S971044