Controlled Ion and Water Uptake

How sodium is taken up during salt stress is not clear. Possibly, monovalent cation channels and transporters serving potassium nutrition could be entry routes for sodium as well (Table 19.1). However, channels of the AKT/KAT type are typically highly selective.72 In contrast, significant influx of sodium through a potassium transporter, HKT in wheat, has been reported.73-74 The HKT transporter from wheat has been described as a K+/ Na+ co-transporter, which mainly transports sodium when the Na+/K+ ratio is high. Preliminary data suggest that the homolog of HKT in rice is predominantly a sodium transporter,75 but it is still unclear whether sodium uptake in planta is accomplished by the HKT type system. Equally unresolved is the cell type in which HKT is present. Based on in situ hybridizations, HKT transcripts have been reported in cortical cells of the root in wheat,73 and expressed in cells of the epidermis and endodermis in rice.75 Definite information will have to wait until we have a better understanding about channel distribution in different cells of the tissues where those transport systems are expressed, as well as detailed analyses of their specificity and contribution to overall potassium and sodium transport. As we approach the time when all genes or putative coding regions are known in several eukary-otes, the necessity for additional information becomes paramount. To completely describe function, detailed knowledge will be essential about cellular localization; tissue, developmental and environmental specificity; and bio-

Table 19.2. Transferred genes affecting plant stress tolerance



Host Species


(N. plumbaginifolia)


N. tabacum M. sativa

Organelle targeted expression for reduced damage by ROS


MtID (E. coli)

Mannitol 1-P DH

N. tabacum A. thaliana N. tabacum

Sodium tolerance at early growth Enhanced germination in NaCI Chloroplast location, ROS scavenging; Calvin-cycle protected


(H. vulgare)


O. sativa

Maintenance of higher growth rate by stressed plants

(ice plant)

myo-inositol O-methyltransterase

N. tabacum

Stress-induced accumulation of D-ononitol

(B. subtilis)


N. tabacum

Fructan accumulation; higher growth during drought stress

(S. cerevisiae)

trehalose synthase

N. tabacum

Low conc. trehalose; increased drought tolerance

(A. globiformis)

choline oxidase

A. thaliana

Glycinebetaine accumulation; enhanced tolerance


Table 19.2., (cont'd) Transferred genes affecting plant stress tolerance



Host Species


(V. aconitifolia)


N. tabacum

Proline accumulation leading to lowering of osmotic potential


FeSOD (A. thaliana)


N. tabacum

PSII/plasma membrane protection/methyl viologen


Cst/Cpx (N. tabacum)


N. tabacum

Increase of oxidized glutathione (GSSG); enhanced seedling growth


While the effects of overexpression indicate protection, the mechanisms leading to enhanced tolerance under controlled growth conditions are largely not understood. High accumulation of mannitol,1 07 or sorbitol1 08 in transgenic tobacco lines reduces growth.

physical and biochemical characteristics of the proteins.

Water channels, aquaporins (AQP), are found in all organisms as members of a superfamily of membrane proteins, 26-30 kDa in size, termed MIP (major intrinsic protein).76-77 The presence of aquaporins increases membrane permeability to water in both directions, depending on osmotic pressure differences across the membrane. Some family members encode glycerol facilitators. Other MIPs in microorganisms, animals and plants may mediate ion transport and/or the movement of small non-electrolytes, such as urea.77-78

In vertebrate animals it seems that at most 10 MIP genes are present. They are expressed in different tissues, most highly in erythrocytes, kidney cells and the brain. In contrast, Arabidopsis contains at least 23 MIP-like coding regions,79 and in corn the number of sequences homologous to Mip transcripts is at least 31.80 Sequence signatures of the Arabidopsis MIP indicate two large subfamilies of 10 to 12 proteins each, whose members are either plasma membrane-located (PIP) or tonoplast-located (TIP) and one MIP diverging from the others which has not been characterized in detail.79 While some of the genes might encode facilitators for diverse small metabolites or ions, eight MIP proteins have already been identified as aquaporins. Why are there so many plant aquaporins? There are several possibilities which might explain the high number.

We will discuss three possibilities only briefly and the fourth in more detail. First, MIP-intrinsic functional variations might allow for AQP to be active at different membrane osmotic potentials. Second, func- tional differences could have evolved for fine tuning water flux through the plant—with high conductance AQP located in the root cortex and vascular tissues which accommodate bulk fluxes and low conductance channels between mesophyll cells, for example, or even within the cytosol and organelles and the vacuole. Third, cell-specific differences in accommodating water flux, not water transport per se, would determine gene number— requiring different promoters, alterations in RNA stability and translation and protein half life regulation.

This explanation is similar to the following, and both find precedence in the presence of, for example, a large number of genes encoding plasma membrane H+-ATPases, AHA, which are differentially expressed throughout the plant.81-82

Last, MIP duplication and diversification could have been dictated by the need for a flexible response to environmental changes in water availability, demanding the presence of several sets of AQP. This assumes evolution of one set of AQP genes for stress responses, and that this set is different from others. It is conceivable that Mip genes exist (set 1) that take care of cell expansion following meristem-atic activity—and this function (missing from animals) might require regulatory circuits separate from those necessary in genes that perform housekeeping (set 2, also found in animals) and stress-response function (set 3). Alignments of sequences indicate that within the PIP and TIP classes, subfamilies of two to four closely related sequences exist,78-79,83 which might represent the three sets of genes. These subfamilies can be inferred because of sequence clustering within the alignment tree that unites sequences from plant orders and genera widely separated during evolution. Although we do not have enough data with respect to AQP protein expression, cell specificity and regulation during stress, it may eventually become possible to predict function by position in an evolutionary tree. Several MIP associated with cell expansion, developmental specificity and stress functions have been described.

One example from our own work is presented in Figure 19.1. The promoter for the ice plant plasma membrane aquaporin MIPB has previously been expressed in tobacco. Its activity has been monitored by GUS expression.84 The MIPB promoter was also inserted into Arabidopsis to control the expression of luciferase. In homozygous lines, luciferase expression has been monitored in response to various stresses and chemical agents that interfere with signal transduction. In Arabidopsis, this ice plant promoter is induced only by drought conditions and by elevated calcium.85 Sodium stress and osmotic stress by mannitol had no effect. Figure 19.1 compares the luciferase signal under normal growth conditions (left panel) in Arabidopsis seedlings with the signal after a 20 minute drought period (after the seedlings had been removed from the agar plate). Within the 20 minutes, signal strength increased more than 10-fold. Such experiments provide useful information about stress responses and shed light on signaling cascades. In addition, the heterologous promoter could become useful as an expression control element in transgenic engineering projects.

Most important for our discussion is how MIP gene expression, protein amount and aquaporin activity are controlled during development and under environmental stress. Regulation is by gene expression and protein amount, and possibly also by posttranslational modification; but as yet we have little information on mechanistic details in plants. Weig et al79 used quantitative PCR amplification for the 23 Arabidopsis MIP and found differences in mRNA amounts spanning several orders of magnitude. Differences in RNA amounts for each MIP in roots, leaves, bolts, flowers and siliques were equally pronounced. No signals were detected for at least three MIP, suggesting that these might be expressed under conditions not found during normal growth or that they are expressed in only a few cells or at very low levels. For several MIP in the ice plant, stress-altered mRNA amounts have been observed (see Maurel77 for a review).83,86 AQP expression also responds to drought and low temperature, hormone treatment (ABA, cytokinine, GA), light and pathogen infection.77-78,87-88 Once all genes are known, the analysis of such a large gene family can best be done by in situ hybridization, immunocyt-ology with specific antibodies and analysis in ordered DNA microarrays. Employing these techniques together, the amount, location and regulation of the genes during development and under different environmental conditions can be known.

The discovery and preliminary characterization of AQP in plants has provided more questions than answers. Their existence cannot be questioned, and many MIP are water channels. It is then intuitively obvious that control over their action is important under stress conditions. Although there are few data available, it is equally clear that regulation during stress is complex, involving transcrip-

tional and posttranscriptional controls which seem to involve synthesis, membrane traffic and reversible insertion into membranes, complex assembly and MIP protein half life.

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