Tetraploidy enhances the ability to exclude chloride from leaves in carrizo citrange seedlings.

Tetraploid citrus seedlings are more tolerant to salt stress than diploid genotypes. To provide insight into the causes of differences in salt tolerance due to ploidy and thus to better understand Cl- exclusion mechanisms in citrus, diploid and tetraploid seedlings of Carrizo citrange (CC) were grown at 0 (control) and 40mM NaCl (salt-treated) medium for 20 days. Chloride uptake and root-to-shoot translocation rates were on average 1.4-fold higher in diploid than in tetraploid salt-treated plants, which resulted in a greater (1.6-fold) Cl- build up in the leaves of the former. Root hydraulic conductance and leaf transpiration rate were 58% and 17% lower, respectively, in tetraploid than in diploid control plants. Differences remained after salt treatment which reduced these parameters by 30-40% in both genotypes. Morphology of the root system was significantly influenced by ploidy. Tetraploid roots were less branched and with lower number of root tips than those of diploid plants. The cross-section diameter and area were lower in the diploid, and consequently specific root length was higher (1.7-fold) than in tetraploid plants. The exodermis in sections close to the root apex was broader and with higher deposition of suberin in cell walls in the tetraploid than in the diploid genotype. Net CO2 assimilation rate in tetraploid salt-treated seedlings was 1.5-fold higher than in diploid salt-treated plants, likely due to the loss of photosynthetic capacity of diploid plants induced by Cl- toxicity. Leaf damage was much higher, in terms of burnt area and defoliation, in diploid than in tetraploid salt-treated plants (8- and 6-fold, respectively). Salt treatment significantly reduced (37%) the dry weight of the diploid plants, but did not affect the tetraploids. In conclusion, tetraploid CC plants appear more tolerant to salinization and this effect seems mainly due to differences in morphological and histological traits of roots affecting hydraulic conductance and transpiration rate. These results may suggest that tetraploid CC used as rootstock could improve salt tolerance in citrus trees.

Expression profiling of two stress-inducible genes encoding for miraculin-like proteins in citrus plants under insect infestation or salinity stress.

The expression of two genes, namely Mir1 and Mir3 and the abundance of their encoded proteins, the putative miraculin-like proteins, MLP1 and MLP3, showing similarity to the Kunitz family of protease inhibitors, were monitored in the leaves of the citrus variety, 'Clementine' after Tetranychus urticae infestation and elicitor treatments, or in the leaves of three other diploid citrus: 'Willow leaf', 'Cleopatra' mandarins and 'Trifoliate' orange, as well as their respective doubled diploids and the allotetraploid somatic hybrid 'FLHORAG1' under salt stress. RT-PCR and 2-DE indicated that Mir1 and Mir3 and their products were present at low-basal expression in all citrus genotypes. Both genes and products were induced in the 'Clementine' leaves infested by T. urticae, but a contrasting profile was observed under elicitor treatments. Under salt stress, the two genes showed an expression pattern contrasting each other and depending on the genotypes. 'Cleopatra' mandarin, 'Trifoliate' orange and 'FLHORAG1' presented overexpression of Mir3 and MLP3 and decreased levels of Mir1 and MPL1. The opposite behaviour was found in 'Willow leaf' mandarin. The positive correlation of the expression profile of the two genes with that of a gene encoding a putative apoplastic cysteine protease (CysP) might suggest a possible interaction of the respective encoded proteins during the response to biotic stress. Under salt stress, CysP and Mir 1 showed a similar expression pattern but only at transcript level. The possible occurrence of post-translational CysP regulation is discussed.

Comparative analysis of proteome changes induced by the two spotted spider mite Tetranychus urticae and methyl jasmonate in citrus leaves.

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis- and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.

A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: a follow up.

With the aim of studying differentially expressed proteins as a function of abiotic and biotic stress in citrus plants, we optimized a protocol for the extraction of total leaf proteins and their 2-DE separation using commercially available immobilized pH gradient strips (IPGs) in the first dimension. Critical factors for good reproducibility of citrus leaf protein separation were identified: trichloroacetic acid (TCA)/acetone precipitation after extraction in lysis buffer, sample fractionation on narrow range overlapping IPGs and sample-cup loading at the anodic or cathodic end of the strip. The use of thiourea and a strong detergent (C7BzO) in the solubilization/rehydration buffer, coupled with the increase to 10% of SDS in the equilibration buffer before the second dimension seemed to affect positively the resolution of basic proteins. Using our protocol we resolved about 30 basic proteins on 6.3-8.3 pH range strips. Further, our protocol was successfully applied reproducibly on the analysis of control and salt exposed leaf samples of Citrus reshni Hort. Ex Tan.

An aquaglyceroporin is abundantly expressed early in the development of the suspensor and the embryo proper of loblolly pine.

In contrast to angiosperms, pines and other gymnosperms form well-developed suspensors in somatic embryogenic cultures. This creates a useful system to study suspensor biology. In a study of gene expression during the early stages of conifer embryogenesis, we identified a transcript, PtNIP1;1, that is abundant in immature loblolly pine (Pinus taeda) zygotic and somatic embryos, but is undetectable in later-stage embryos, megagametophytes, and roots, stems, and needles from 1 year-old seedlings. Analysis of PtNIP1;1 transcript in embryo proper and suspensor tissues by reverse transcription-polymerase chain reaction suggests preferential expression in the suspensor. Based on comparisons of derived amino acid sequences, PtNIP1;1 belongs to the nodulin-like members of the major intrinsic protein superfamily branch of the aquaporin (major intrinsic protein) superfamily. Through heterologous expression in Xenopus laevis oocytes and the yeast (Saccharomyces cerevisiae) fps1(-) mutant, PtNIP1;1 has been shown to be an active aquaglyceroporin.

The role of ABA and the transpiration stream in the regulation of the osmotic water permeability of leaf cells.

The transpiration stream that passes through a plant may follow an apoplastic route, with low resistance to flow, or a cell-to-cell route, in which cellular membranes impede water flow. However, passage of water through membranes can be facilitated by aquaporins thereby decreasing resistance. We investigated the relationship between transpiration, which can be down-regulated by abscisic acid (ABA) or by high humidity, and the osmotic water permeability (P(os)) of protoplasts. By using leaf protoplasts of wild-type (wt) Arabidopsis thaliana plants and of mutants that are low in ABA (aba1) or insensitive to ABA (abi1 and abi2), we found that protoplasts from aba1 and abi mutants have very low P(os) values compared with those from wt plants when the plants are grown at 45% relative humidity. High values of P(os) were found 3 h after the addition of ABA to the culture medium of aba1 plants; addition of ABA to abi plants did not restore the P(os) to wt levels. There was no such increase in P(os) when excised leaves of aba1 plants were treated with ABA. When the transpiration stream was attenuated by growing the plants at 85% relative humidity, the P(os) of protoplasts from all plants (wt and mutants) was higher. We suggest that attenuation of the transpiration stream in whole plants is required for the up-regulation of the P(os) of the membranes, and that this up-regulation, which does not require ABA, is mediated by the activation of aquaporins in the plasma membrane.

Brassinolide may control aquaporin activities in Arabidopsis thaliana.

It is usually assumed that aquaporins present in the cellular membranes could be an important route in the control of water flux in plants, but evidence for this hypothesis is scarce. In this paper, we report measurements of the osmotic permeability (P(os) of protoplasts isolated from hypocotyls of wild-type and mutant Arabidopsis thaliana (L.) Heynh. Mutants were affected in their growth and exhibited different sensitivities to the phytohormone, brassinolide. For the two mutants studied (cpd: constitutive photomorphogenesis and dwarfism; bril: brassinosteroid insensitive), hypocotyl length was correlated to P(os) for the protoplasts. Under experimental conditions where hypocotyl growth had ceased, restoration of root, hypocotyl and petiole growth by brassinolide was correlated with an increase in P(os) of the hypocotyl protoplasts. We consider that the increase in Pos of the hypocotyl cells was needed because these cells were part of the transcellular water pathway of the plant. This is the first time, to our knowledge, that brassinolide has been shown to be involved in the modification of the water-transport properties of cell membranes. Our results also emphasize the importance of aquaporins and the transcellular pathway in water transport under normal growth conditions.

Water permeability and revolving movement in Phaseolus vulgaris L. twining shoots.

Osmotic water permeability (Pos) was measured in protoplasts isolated from different tissues of Phaseolus vulgaris twining shoot. Parenchyma protoplasts exhibited more frequently high Pos values than epidermis protoplasts did. Water channels could facilitate water movement between parenchyma cells whereas cell-to-cell water transport mostly occurs through plasmodesmata in epidermis.

Osmotic water permeability of isolated vacuoles

We measured the osmotic water permeability (P(os)) of vacuoles isolated from onion (Allium cepa L.), rape (Brassica napus L.), petunia (Petunia hybrida Hook.) and red beet (Beta vulgaris L.). For all the vacuolar types investigated, P(os) values were in the range 200-1000 &mgr;m s(-1). The change in membrane surface area induced by an osmotic gradient was smaller than 2-6%. The vacuolar P(os) values for red beet and onion were reduced by 1 mM HgCl(2), to 14% and 30% of the control values, respectively, but were partially restored to 51% and 76% by 5 mM beta-mercaptoethanol. These results suggest that aquaporins were present in all the vacuoles tested. In HgCl(2)-treated onion vacuoles, the reduced P(os) (56 &mgr;m s(-1)) had a low activation energy (approx. 6 kJ mol(-1)), indicating that water permeation was still occurring mainly via aquaporins, and that the water permeability of the lipid part of the vacuolar membrane is probably very low.

Osmotic water permeability of isolated protoplasts. Modifications during development

A transference chamber was developed to measure the osmotic water permeability coefficient (Pos) in protoplasts 40 to 120 µm in diameter. The protoplast was held by a micropipette and submitted to a steep osmotic gradient created in the transference chamber. Pos was derived from the changes in protoplast dimensions, as measured using a light microscope. Permeabilities were in the range 1 to 1000 µm s-1 for the various types of protoplasts tested. The precision for Pos was