Physiologia Plantarum

Portillo, Mary; Fenoll, Carmen; Escobar, Carolina

doi: 10.1111/j.1399-3054.2006.00716.xpmid: N/A

Highly sensitive techniques for transcriptome analysis, such as microarrays, complementary DNA‐amplified fragment length polymorphisms (cDNA‐AFLPs), and others currently used in functional genomics require a high RNA quality and integrity, as well as reproducibility among extractions of replicates from the same tissue. There are, however, few technical papers comparing different homogenization techniques and reagents to extract RNA from small quantities of plant tissue. We extracted RNA from tomato seedlings with the three different commercial reagents TRIZOL LS®, TRIZOL®, and TRI Reagent® in combination with pulverization, homogenization‐maceration in a mortar, and homogenization with mild vibration plus glass beads, and evaluated total RNA integrity‐quality and yield. Pulverization under liquid nitrogen combined with TRIZOL LS® as extraction reagent and homogenization‐maceration in mortar with TRI Reagent®, are the procedures that rendered higher RNA yield, integrity and quality, as well as reproducibility among independent RNA extractions. In contrast, short mild vibration pulses (4500 r.p.m. for 5 s) mixed with glass beads, rendered low extraction efficiency and caused, in most cases, partial RNA degradation.

Tan, Fui‐Ching; Swain, Steve M.

doi: 10.1111/j.1399-3054.2006.00724.xpmid: N/A

Flowering is an integral developmental process in angiosperms, crucial to reproductive success and continuity of the species through time. Some angiosperms complete their life cycle within a year (annual plants), and others have a longer reproductive life, which is characterized by the generation of new flowering and vegetative shoots every year (perennial plants). Despite the differences in their lifespan, the underlying genetics of flower induction and floral organ formation appears to be similar among these plants. Hence, the knowledge gained from the study of flowering mechanism in Arabidopsis thaliana can be used to better understand similar processes in other plant species, especially the perennials, which usually have a long generation time and are not amenable to genetic analysis. Using Arabidopsis as a model, we briefly discuss the current understanding of the transition from vegetative to reproductive growth and the subsequent formation of individual floral organs, and how this knowledge has been successfully applied to the identification of homologous genes from perennial crops. Although annuals appear to share many similarities with perennials in terms of gene function, they differ in their commitment to flowering. Once an annual reaches the reproductive phase, all meristems are typically converted into either floral or inflorescence meristems. In contrast, each year, each meristem of a mature perennial has the choice to produce either a vegetative or a reproductive shoot. The physiology and genetics of flowering in Citrus are used to highlight the complexity of reproductive development in perennials, and to discus possible future research directions.

Immerzeel, Peter; Eppink, Mieke M.; De Vries, Sacco C.; Schols, Henk A.; Voragen, Alphons G. J.

doi: 10.1111/j.1399-3054.2006.00712.xpmid: N/A

Cell wall extracts from a carrot cell culture and tap roots were obtained by sequential extraction with water, EDTA buffer solution and cold sodium hydroxide solution. Arabinogalactan proteins (AGPs) were isolated from the extracts and from the medium of the cell culture and analysed for their molecular weight distribution and carbohydrate composition. Copper ions were used to separate the Yariv positive fractions into AGP fractions with a high and a low level of galacturonic acid (GalA). The GalA rich AGP fractions were incubated with pectin methylesterase and polygalacturonase. This enzyme incubation released GalA fragments from the AGP fractions as monitored by HPAEC and MALDI‐TOF MS. At least part of carrot AGPs from the medium and cell walls may be covalently linked to pectin containing a homogalacturonan structural element.

Vasconsuelo, Andrea; Picotto, Gabriela; Giuletti, Ana M.; Boland, Ricardo

doi: 10.1111/j.1399-3054.2006.00717.xpmid: N/A

We have previously shown that chitosan stimulates anthraquinone synthesis in Rubia tinctorum L. cells through activation of the PLC\PKC, PI3K, MAPK and Ca2+ messenger systems. In view of this evidence, we have now investigated whether guanine nucleotide‐binding G‐proteins are part of the signal transduction mechanism which mediates the elicitor action. The G‐protein agonists mastoparan, AlF4– and GTPyS increased anthraquinone levels to the same extent as chitosan. No additive effects were observed when cultured R. tinctorum cells were treated with agonist and the elicitor together. In agreement with these observations, the G‐protein antagonists suramin and GDPβS abolished the increase in anthraquinone synthesis induced by chitosan. Furthermore, elicitation was not affected in the presence of pertussis toxin. Consistent with this result, when cell cultures were preincubated with a monoclonal anti‐Gαq\11 antibody, the chitosan‐dependent increase in anthraquinone levels was fully inhibited. Moreover, the presence of an immunoreactive protein of the expected size for Gαq\11 (42 kDa) was observed in R. tinctorum microsomal membranes by Western blot analysis using the same antibody. These results indicate that chitosan stimulates anthraquinone synthesis in R. tinctorum cells through a heterotrimeric G‐protein, most likely belonging to the Gαq family.

Monteoliva, Mariela; Guzzo, Carla; González, Claudio A.; Kenis, Juana D.

doi: 10.1111/j.1399-3054.2006.00725.xpmid: N/A

Regulation of nitrate reductase (NR; EC 1.6.6.1) breakdown, measured as loss of maximal activity (MNRA), was studied in leaf segments of 7‐day‐old oat plants in the light for up to 4 h. In segments floating on 1 mM tungstate, NR lost more than 40% of its initial maximal activity. Cycloheximide, high (300 mM) glucose (Glc) and inhibitors of cysteine proteases stabilized NR in situ, suggesting that MNRA decrease was due to the hydrolysis of NR by a short‐lived, glucose‐modulated cysteine protease. Loss of MNRA was accelerated by cantharidin (CTHR) and inhibited by staurosporine, suggesting that NR breakdown required continuous phosphorylation. High glucose inhibited any further MNRA decrease when supplied after a 30‐min pretreatment with CTHR, suggesting that a phosphorylated protein was its target. Isoosmolar polyethylene glycol also stabilized NR but not in the presence of CTHR. Low (30 mM) Glc stabilized NR only in the presence of Ca2+, and CTHR inhibited its effect. EGTA and LaCl3 completely arrested the effects of both high‐ and low‐ Glc. Like low D‐Glc, low L‐Glc (glucose analog not transported) inhibited NR breakdown in the presence of Ca2+, but at high concentration only 2‐deoxyglucose, that is phosphorylated but not further metabolized, and glucose‐6P were effective in the presence of CTHR, suggesting that receptors for high‐ and low‐ Glc were located in different cell compartments. It is proposed that high‐ and low‐ Glc trigger different signalling pathways, with calcium as a common upstream secondary messenger and protein kinases and protein phosphatases being downstream components in the cascade of reactions that modulates NR proteolysis.

Li, Yujing; Heaton, Andrew C. P.; Carreira, Laura; Meagher, Richard B.

doi: 10.1111/j.1399-3054.2006.00732.xpmid: N/A

Arsenic and mercury are among the most toxic elemental pollutants in the environment, endangering human health and ecological integrity. Both elements are found in highly thiol‐reactive forms, arsenite and Hg(II), respectively, in plant tissues. Overexpression of Escherichia coliγ‐glutamylcysteine synthetase (ECS) or glutathione synthetase (GS) in Arabidopsis thaliana plants provided significant increases in the thiol peptides glutathione (GSH) and γ‐glutamylcysteine (γ‐EC), and/or phytochelatins (PCs), and some resistance to arsenic and mercury, but no substantial increases in the levels of these elements in above‐ground tissues. In contrast, the co‐expression of ECS and GS in ECS × GS lines produced significant increases in tolerance to toxic levels of mercury. The ECS × GS co‐expression line accumulated 35‐fold more biomass and three‐fold more mercury aboveground than the wild type (WT) when grown on Hg(II). No increases in arsenic accumulation were detected in the ECS × GS line. Increased resistance to and accumulation of mercury apparently resulted from enhanced root concentrations of PCs in ECS × GS co‐expression lines not seen in the wild type or lines expressing ECS or GS alone. Correlations between the levels of arsenic and mercury resistance and accumulation and increases in the accumulation of the various thiol peptides in the ECS, GS and ECS × GS transgenic plant lines are discussed.

Liu, Hong‐Yan; Sun, Wei‐Ning; Su, Wei‐Ai; Tang, Zhang‐Cheng

doi: 10.1111/j.1399-3054.2006.00709.xpmid: N/A

Aquaporins and potassium channel proteins are probably critical for a plant to maintain proper cytosolic osmolarity in response to drought or other stresses. However, evidence linking water channel and potassium channel functions in plants remains to be demonstrated. The present study examined K+ channel/transporters and water channels in rice (Oryza sativa L. spp. indica cv. Guangluai 4) to reveal a potential functional correlation. The mRNA expression levels of plasma membrane intrinsic proteins (PIPs) and K+ channel/transporters responded similarly to K+ starvation or water deprivation. Transcription of the PIP‐ and K+ channel‐encoding genes was induced by K+ starvation and could be downregulated by polyethylene glycol (PEG)‐mediated water deficit. Consistent with the induced PIP expression, root hydraulic conductivity (Lp) also increased during K+ starvation. Furthermore, the K+ uptake capacity, but not the K+ content, was probably influenced by K+ starvation. Caesium chloride treatment decreased K+ content in the rice seedlings and reduced root Lp as did mercuric chloride. These results are compatible with the conclusion that PIP and K+ channel/transporters are functionally co‐regulated in rice osmoregulation.

D'Haese, David; Horemans, Nele; De Coen, Wim; Guisez, Yves

doi: 10.1111/j.1399-3054.2006.00711.xpmid: N/A

To better understand the response of a plant to O3 stress, an integrated microarray analysis was performed on Arabidopsis plants exposed during 2 days to purified air or 150 nl l−1 O3, 8 h day−1. Agilent Arabidopsis 2 Oligo Microarrays were used of which the reliability was confirmed by quantitative real‐time PCR of nine randomly selected genes. We confirmed the O3 responsiveness of heat shock proteins (HSPs), glutathione‐S‐tranferases and genes involved in cell wall stiffening and microbial defence. Whereas, a previous study revealed that during an early stage of the O3 stress response, gene expression was strongly dependent on jasmonic acid and ethylene, we report that at a later stage (48 h) synthesis of jasmonic acid and ethylene was downregulated. In addition, we observed the simultaneous induction of salicylic acid synthesis and genes involved in programmed cell death and senescence. Also typically, the later stage of the response to O3 appeared to be the induction of the complete pathway leading to the biosynthesis of anthocyanin diglucosides and the induction of thioredoxin‐based redox control. Surprisingly absent in the list of induced genes were genes involved in ASC‐dependent antioxidation, few of which were found to be induced after 12 h of O3 exposure in another study. We discuss these and other particular results of the microarray analysis and provide a map depicting significantly affected genes and their pathways highlighting their interrelationships and subcellular localization.

Zhu, Guo‐Yu; Geuns, Jan M. C.; Dussert, Stéphane; Swennen, Rony; Panis, Bart

doi: 10.1111/j.1399-3054.2006.00713.xpmid: N/A

To understand the mechanisms of sucrose‐induced acclimation in relation to plant cryopreservation, sugars, sterols, fatty acids of different lipid fractions (neutral lipids, glycolipids and sphingolipids and phospholipids), as well as free fatty acids were analyzed in proliferating meristem cultures of different banana varieties. The four banana varieties that were selected show different post‐thaw shoot regeneration rates (0–53.4%). All mentioned parameters were analyzed using (1) control meristems that were cultured on a normal sucrose concentration (0.09 M), which resulted in low survival after cryopreservation; and (2) 2‐week sucrose precultured meristems (0.4 M). This sucrose preculture, essential for regeneration after cryopreservation, resulted in a significant increase of each of seven sugars detected. The ratio of stigmasterol/sitosterol (St/Si) in sucrose‐pretreated meristems significantly increased. The sucrose pretreatment also resulted in a significant increase of total fatty acid content of the neutral lipid fraction and of the glycolipid and sphingolipid fraction, as well as the total free fatty acid content. The individual fatty acid content of the phospholipids was differently changed by the sucrose pretreatment for the given varieties studied. In most cases, sucrose pretreatment resulted in an increase of the double bond index (DBI) in the neutral lipids and a decrease of DBI in the glycolipids and sphingolipids, in phospholipids as well as in free fatty acids. Principal component analysis of all collected data revealed that (1) for the control material, sucrose and total sugar contents were closely linked to the post‐thaw shoot regeneration, suggesting that sucrose and total sugar may be main limiting factors to survive cryopreservation; (2) accumulation of large quantities of sugars (glucose, fructose, sucrose and total sugar) in sucrose‐pretreated material cannot explain the differences in survival after cryopreservation of the four banana varieties. We assume that a minimal amount of sugars is needed in meristem cultures to survive cryopreservation. Still, other limiting factors do influence the survival following the sucrose pretreatment. We observed that the parameters which are closely linked to the post‐thaw shoot regeneration are a minimal change in the ratios of St/Si, the minimal change of the DBI of phospholipids and free fatty acids, as well as linoleic acid content (C18:2); and (3) inositol, raffinose, myristic acid (C14:0) and oleic acid (C18:1) were present in small quantities; however, they could be correlated to survival after cryopreservation, suggesting that they may be also involved in cryopreservation process.

Mitsuya, Shiro; Taniguchi, Mitsutaka; Miyake, Hiroshi; Takabe, Tetsuko

doi: 10.1111/j.1399-3054.2006.00714.xpmid: N/A

We have investigated whether the overexpression of RCI2A gene causes an enhanced salt‐tolerant phenotype in Arabidopsis thaliana. Although the growth of RCI2A‐overexpressing transgenic plants was comparable with that of wild type under normal conditions, high salinity treatment caused decreased accumulation of Na+ and ameliorated suppression of the shoot growth of transgenic plants than that of wild type. Under high salinity treatment, the chlorophyll content of the shoots of wild‐type plants significantly decreased compared with transgenic plants. The increases of malondialdehyde (MDA) and of H2O2 production caused by high salinity were greater in the shoots of wild type than in that of transgenic plants. These results suggest that overexpression of RCI2A can alleviate salinity‐induced growth suppression and photooxidative damages via reducing Na+ uptake into the shoots.