Journal of Experimental Botany

JXB Advance Access originally published online on November 1, 2005
Journal of Experimental Botany 2006 57(5):1137-1147; doi:10.1093/jxb/erj001

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RESEARCH PAPER

The role of monovalent cation transporters in plant responses to salinity

Frans J. M. Maathuis^*

Department of Biology, University of York, PO Box 373, York YO10 5YW, UK

^* Fax: +44 (0)1904 328666. E-mail: fjm3{at}york.ac.uk

Received 28 June 2005; Accepted 31 August 2005

	Abstract

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
Exposure to high ambient levels of NaCl affects plant water relations and creates ionic stress in the form of the cellular accumulation of Cl^– and, in particular, Na⁺ ions. However, salt stress also impacts heavily on the homeostasis of other ions such as Ca²⁺, K⁺, and and therefore requires insights into how transport and compartmentation of these nutrients is altered during salinity stress. A genomics approach can greatly help with the identification of genes, and therefore potentially gene products, that are involved in plant salinity. Both the literature and public databases contain the results of many genomics studies and, in this report, those data are collated in the context of cation membrane transport and salinity. The efficacy of genomics approaches in isolation is low due to large inherent variability and the exclusion of gene products that are predominantly regulated post-transcriptionally. In conjunction with complementary approaches, however, transcriptomics can help identify important transcripts and relevant associations between physiological processes. This analysis identified (i) vascular K⁺ circulation, (ii) root shoot translocation of Ca²⁺, and (iii) transition metal homeostasis as potentially important aspects of the plant response to salt stress.

Key words: Cation transport, microarray, salinity stress, transcriptomics

	Introduction

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
Plant mineral nutrition is crucial to plant growth and development and, as a consequence, to agriculture and human health. However, optimal plant growth is rarely achieved in non-agricultural settings since most soils show deficiency for one or more essential minerals, which may lead to nutrient stress. In addition, high concentrations of inorganic ions can further compromise plant growth and development which is particularly the case in environments suffering from high levels of salinization.

Many of the detrimental effects of salinity stress have been characterized both at the whole plant and at the cellular level. The rapid onset of osmotic stress leads to water deficit, which, at the level of nutrition, has several consequences. Reduced transpiration has detrimental effects on the long-distance transport of low mobility ions such as Ca²⁺, thus creating Ca²⁺ deficiency, particularly in fast-growing tissues like expanding leaves. Restoration of water relations typically requires the uptake of osmoticum in the form of Cl^– and Na⁺ ions. These are predominantly stored in the vacuole with a concomitant loss of cellular K⁺.

Apart from rapidly occurring water stress, increases in ambient salt concentrations can lead to toxic accumulation of ions such as Cl^– and, in particular, Na⁺ in the cytosol. The disproportionate presence of Na⁺ in both cellular and extracellular compartments negatively impacts on the acquisition and homeostasis of essential nutrients such as K⁺ and Ca²⁺. The negative effects find their origin in many phenomena: ionic interactions between Ca²⁺ and cellular components such as cell wall pectins and membrane phospholipids are sensitive to excess cations such as Na⁺. Although these sites typically have a much higher affinity for Ca²⁺ than for Na⁺ the large molar Na⁺:Ca²⁺ ratio leads to dissociation of Ca²⁺ from its binding sites, affecting the integrity of cell walls and cell membranes. In the cytosol, the presence of K⁺ is essential for the activation of many enzymes, for example, those involved in pyruvate synthesis and protein translation. Due to physicochemical similarities between Na⁺ and K⁺, excess Na⁺ will tend to substitute K⁺ for Na⁺ at these binding sites and hence impair cellular biochemistry. A third mechanism affected by high levels of salinity is nutrient transport itself. Specific transport systems have been shown to be inhibited by the presence of large amounts of ions such as Cl^– and Na⁺. These include high affinity K⁺ transporters of the KUP family (Santa-Maria et al., 1997) and transporters (Cerezo et al., 1997).

Significant alterations in transporter transcript level during salinity stress may point to potential roles of these gene products and/or their substrates during plant salinity stress. The use of microarrays to probe transcriptomes has been employed for several years and many reports are available with particular reference to alterations in transcriptomes during salinity stress. In addition to data published in the literature, there are now data available to the public. The aim of this report is to classify and integrate these generic results with tissue and cell-specific expression data and data regarding specific cation transporters in order to identify those cation transporters that are most likely to play a significant role in plant salinity tolerance.

	Materials and methods

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
Transcriptomics analyses
Public data were derived from the Genevestigator site (https://www.genevestigator.ethz.ch) and published reports. Data for mature Arabidopsis thaliana (L.) ecotype Columbia shoot tissue from salt-treated plants were derived from experimentation using AMT arrays as described in Maathuis et al. (2003).

Growth assays and ion contents analysed
For growth assays, germination and plant growth were on standard medium (Maathuis et al., 2003) for 6–7 d. Plants were subsequently transferred to plates containing standard medium or standard medium supplemented with 80 mM NaCl. Growth rates were determined by measuring seedling fresh weight after 5 d growth, relative to control conditions. For Na⁺ content measurements, mature plants were exposed to growth medium supplemented with 80 mM NaCl for 72 h. Plants were washed twice for 10 min in ice-cold 20 mM CaCl₂ to remove cell wall Na⁺, weighed, and dried at 80 °C for 72 h. Ion analysis was carried out on acid-extracted root and shoot tissue using ICP analysis. For all experiments 10–12 mature plants were used and at least three independent replicates were carried out.

	Results and discussion

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
Setting the boundaries
Most of the available data inevitably pertain to A. thaliana and the genomics of this model species will provide the bulk of both the results and their discussion. However, where possible, data from other species are also used. Around 550 transcripts were included for transcriptomics analyses and these were limited to those known or likely to play a role in the transport of the inorganic cations. Thus, the cation transport families included were: K⁺ channels (AKT/KAT/SKOR/KCO); non-selective channels (CNGC/GLR/TPC); K⁺ transporters (KUP/HAK/KEA/HKT); Ca²⁺, Mg²⁺, Na⁺, and cation proton antiport (CAX/MHX/CHX/NHX); Ca²⁺ and metal pumps (ACA/HMA); Mg²⁺ transporters (MTG); metal transporters (Nramp/COPT/IRT/ZIP/MTP/YSL); ammonium transporters (AMT); and transcripts that are annotated as transport protein of unknown function.

All the transcripts listed above were scored when salinity induced a 3-fold or larger change in transcript abundance. For completeness, additional cation transporters that are known to function in cation homeostasis during salinity stress were also included in further analyses, irrespective of reported changes in transcript level. The resulting population of annotated and unknown transporters was subsequently analysed with respect to expression patterns, using Genevestigator, MPSS, and published data, and with respect to reported functional data. To discriminate between transcripts that are most likely involved specifically with ionic aspects of salinity stress and those that respond to osmotic stress or generic stress, Table 1 indicates, in addition, whether salinity-regulated transcripts were also affected by either osmotic/drought stress, K⁺ deprivation, or Ca²⁺ deprivation.

View this table: [in this window] [in a new window]   Table 1. Annotated cation and unknown transcripts, identified from a number of sources, that show a 3-fold or more change in abundance in response to salt stress

 
Salinity affects transcription of cation and unknown transporters
Table 1 lists all annotated cation transporters and unknown transporters that show a larger than 3-fold change in transcript abundance during salinity. This list includes around 40 genes that are annotated as cation transporter and 45 unknown genes, from various transporter families. Where data on separate root and shoot tissue and membrane location are available these have been provided. Reported ratios vary roughly from a 20 times reduction to a 50 times increase in transcript level and typically cover salt treatment times from 2–96 h.

Annotated genes and those that have previously been shown to be relevant in plant salt tolerance (i.e. NHX1 and NHX7) can be categorized into a small number of functional classes that are listed in Table 2: (i) Na⁺ transport, (ii) carrier- and pump-mediated heavy metal transport, (iii) K⁺ transport through carriers and channels, (iv) Ca²⁺ transport through pumps and exchange systems, (v) cation exchange, and (vi) monovalent non-selective ion channels. Figure 1 shows expression data for most of the regulated genes based on Genevestigator output. These data are solely derived from comparisons and the averaging of microarray signals, but should, nevertheless, give a good approximation of relative expression levels for different transcripts and tissues.

View this table: [in this window] [in a new window]   Table 2. Functional categorization of annotated transcripts listed in Table 1 extended with transporters that have previously been shown to impact on plant salinity tolerance (NHX1 and NHX7): references are as denoted in Table 1.

 

View larger version (52K): [in this window] [in a new window]   Fig. 1. Relative expression levels in seedlings, inflorescence, rosette leaves, and root tissue of genes listed in Table 1. Expression levels are based on signal intensities from microarrays, collated and available at the Genevestigator site. Dark colours indicate relatively low expression levels whereas lighter colours indicate relatively high expression levels.

 
The role of Na⁺ transporters:
Cytosolic accumulation of Na⁺ is detrimental to many physiological processes. The intra- and intercellular transport of this ion therefore needs to be carefully regulated. HKT1 functions as a potential Na⁺ uptake pathway in both wheat (Laurie et al., 2002) and arabidopsis (Rus et al., 2001). Antisense expression of this transporter in wheat led to a decrease in unidirectional Na⁺ influx providing direct evidence that it functions as a root Na⁺ uptake mechanism. In Arabidopsis, loss of function in HKT1 suppressed the sodium oversensitive (SOS3) phenotype and largely restored growth of the sos3 mutant at low external K⁺. More recently, the notion that the Arabidopsis HKT1 functions in Na⁺ uptake has been challenged (Maser et al., 2002; Berthomieu et al., 2003) and strong evidence was presented that it functions in Na⁺ translocation from root to shoot and vice versa. The same studies showed no difference in HKT1 transcript abundance in response to salt. By contrast, salt-exposed rice plants did show a reduction in HKT1 transcript level (Horie et al., 2001; Golldack et al., 2002) and its down-regulation in glycophytes such as Arabidopsis and rice might participate in reducing Na⁺ influx during salinity.

Members of the NHX family are invariably annotated as Na⁺:H⁺ antiporters but, at least for some isoforms, are likely to transport other monovalent cations as well. The roles of the vacuolar NHX1 and plasma membrane NHX7(SOS1) have been well documented with NHX1 and NHX7(SOS1) both contributing to cytoplasmic Na⁺ extrusion into the vacuole and apoplast, respectively. Reports have shown that both NHX1 (Apse et al., 1999; Yokoi et al., 2002) and NHX7 (Zhu, 2002) transcript levels correlate with salinity stress and overexpression of both isoforms improves salt tolerance. Data in Table 2 derived from mature root tissue also show considerable increase in transcript levels of NHX4. Unfortunately, no functional assays have been carried out with NHX4 so its membrane location and substrate specificity remain to be discovered.

Overall, both the down-regulation of HKT1 and up-regulation of NHX isoforms could greatly contribute to limiting the Na⁺ load on plant tissue, in particular where cytoplasmic Na⁺ contents are concerned.

Salinity and transition metal transport:
Out of the eight genes in Table 2 that are involved in transition metal transport, seven (IRT1, ZIP8, ZIP10, COPT2, YSL5, Nramp1, and MTP1) show a reduction in transcript levels of up to 16 times during salt stress, whereas only one transcript (HMA1) is up-regulated.

IRT1, ZIP8 and ZIP10 are members of the Zrt, Irt-like protein family that transports divalent metals such as Fe²⁺ and Zn²⁺. IRT1 has been shown to be essential for the uptake of iron from the soil and is expressed at the plasma membrane in the external cell layers of the root (Vert et al., 2002). COPT2 is likely to mediate high affinity Cu²⁺ uptake into the cytoplasm (Sancenon et al., 2003) whereas YSL proteins have been suggested to participate in long-distance translocation and partitioning of metals throughout the plant (DiDonato et al., 2004). MTP1 is expressed at the tonoplast and contributes to Zn²⁺ sequestration in the vacuole. Thus, these proteins are all involved in the uptake of transition metals or their cellular and intercellular partitioning. By contrast, HMA1 which belongs to the P-type (type 1B) heavy metal ATPase family, is the only up-regulated transcript. By analogy to its close homologue, HMA2 (Eren and Arguello, 2004), HMA1 most probably functions as a plasma membrane Zn²⁺ efflux mechanism. Apart from IRT1, whose transcript level was also affected by drought stress, regulation of the other metal transporter transcripts is specifically in response to salinity stress.

The collective data suggest that there may be an important link between ionic aspects of salinity stress and transition metal homeostasis. It appears that uptake of transition metals like Fe²⁺, Zn²⁺, and Cu²⁺ is reduced during salinity stress whereas their active extrusion is promoted. The physiological relevance of this is unclear, since little is known about interactions between salinity stress and the requirement for micronutrients such as Fe²⁺, Cu²⁺, and Zn²⁺. However, through substitution of Ca²⁺ by Na⁺, salinity compromises membrane integrity. The latter may lead to an augmented influx of heavy metals, such as Cu²⁺ and Zn²⁺, and thus the requirement to restrict these fluxes through ZIP- and COPT-type transporters and increased extrusion through HMA-like pumps. Alternatively, salinity-induced perturbation of cellular and intracellular Na⁺, K⁺, and Ca²⁺ fluxes may affect general cation homeostasis, including that of transition metals.

Salinity and K⁺ nutrition:
The chemico-physical similarity between Na⁺ and K⁺ generates pronounced effects on plant K⁺ nutrition during salt stress. The turgor function of vacuolar K⁺ is largely replaced by Na⁺ accumulation requiring tonoplast transporters. The inhibitory effect of Na⁺ on K⁺ uptake mechanisms can create K⁺ deficiency and the build-up of cytoplasmic Na⁺ can interfere with the catalytic role that K⁺ plays in many metabolic processes. Plants possess a large number of active and passive transporters for the uptake, compartmentation and long-distance transport of K⁺. Functional data for many of these are still scarce but it has become increasingly evident that membrane location and tissue-specific expression vary greatly. Since salinity stress severely impacts on K⁺ homeostasis in all plant organs, a differential regulation of K⁺ transport according to cell compartment and plant tissue might be expected.

Members of the HAK/KUP/KT family are involved in both low and high affinity K⁺ transport and, although no firm data exist, are presumably localized at different membranes (Banuelos et al., 2002). Transcription of KUP2, which is predominantly expressed in rapidly growing tissues and has been shown to play a role in cell expansion (Elumalai et al., 2002), is decreased in shoots of NaCl-treated plants (Table 2). Its down-regulation may reflect salinity-induced lack of turgor and a general reduction in growth rates. By contrast, KUP6 and KUP11 are both up-regulated during salt stress, as is a maize KUP homologue in root tissue. Transcript abundance of KUP1 and KUP4 homologues was also found to increase in the ice plant during K⁺ starvation and salt exposure (Su et al., 2001). These isoforms are possibly involved in maintaining cytoplasmic K⁺ levels and/or turgor regulation during conditions where external Na⁺ inhibits K⁺ uptake and cellular Na⁺ replaces K⁺. The K⁺ channel AKT2,3 is up-regulated during salinity. AKT2,3 is weakly voltage-dependent and has been postulated to function as a shunt conductance in phloem cells (Marten et al., 1999). Thus, AKT2,3 may be involved in the recirculation of K⁺ through the phloem. This essential part of plant K⁺ homeostasis is likely to be affected, especially in relation to root/shoot partitioning of Na⁺ and K⁺.

By contrast, the outward rectifying channel SKOR is located in the plasma membrane of stelar root tissue where it functions in K⁺ release into the xylem. Clearly SKOR plays an important role in the root–shoot partitioning of K⁺. As is the case for AKT2,3, the gene encoding SKOR is also significantly up-regulated during salt stress (Table 2). Previously, it has been shown that ABA treatment rapidly and significantly decreases SKOR transcript levels (Gaymard et al., 1998). SKOR up-regulation during salinity stress is therefore unlikely to proceed through an ABA-mediated pathway. The substantial up-regulation of both SKOR in roots and AKT2,3 in shoots during salinity stress would result in increased rates of K⁺ circulation through the vascular tissues and points to a long-distance redistribution of K⁺ between the root and shoot. Interestingly, the opposite, i.e. down-regulation of SKOR and AKT2,3 occur during K⁺ deprivation (Maathuis et al., 2003; Pilot et al., 2003).

A second K⁺ channel, TPK5 (KCO5), is expressed at the tonoplast (K Czempinski, personal communication). No specific data regarding its selectivity and gating characteristics are available, but this channel, in analogy with TPK1 (FJM Maathuis, unpublished results) and TPK4 (Becker et al., 2004), is likely to be K⁺ selective and largely voltage independent and may therefore function in bidirectional trans-tonoplast K⁺ flux. Thus, its decrease in transcript abundance may signify involvement in maintaining cytoplasmic K⁺ levels and/or the exchange of vacuolar K⁺ for Na⁺.

Interactions between salinity stress and Ca²⁺ transport:
Ameliorating effects of externally applied Ca²⁺ on salt stress have been described for many plant species. In addition, it has become established that adaptation of plants to environmental stress requires the regulation of intracellular Ca²⁺ levels. Some of these processes can now be explained mechanistically at the molecular level. For example, through the inhibitory effect of external Ca²⁺ on non-selective ion channels believed to contribute directly to Na⁺ uptake (Maathuis and Sanders, 2001) or through Ca²⁺-dependent signalling during salinity stress (Zhu, 2002). By contrast, salt and, in particular Na⁺, can negatively affect plant Ca²⁺ relations. Some of the structural functions Ca²⁺ plays can be compromised due to the presence of large amounts of Na⁺ which can replace electrostatically bound Ca²⁺ in cell walls and cell membranes. In addition, Ca²⁺ nutrition can be deregulated since the osmotic effects of salinity stress lead to a reduction in transpiration, particularly affecting relatively immobile ions such as Ca²⁺ in their root–shoot translocation.

Expression levels of the vacuolar P-type Ca²⁺ pump ACA4 (Geisler et al., 2000) are down in roots after salt stress. Typically, pumps such as ACA4 remove Ca²⁺ from the cytoplasm to sequester it into the vacuole, ER, or apoplast. In contrast to ACA4, both ACA12 and ACA13 and a maize ACA10 homologue are up-regulated in roots, to more than 30-fold. The latter are all IIB-type ACA isoforms and are believed to express at the plasma membrane rather than endomembranes (Geisler et al., 2000).

Salt stress can lead to decreased influx and reduced xylem loading of Ca²⁺ (Halperin et al., 1997) creating the requirement to maximize (re)translocation of Ca²⁺ to shoot tissue. Xylem loading of Ca²⁺ has been suggested to be largely apoplastic (White, 2001). Therefore, a concomitant reduction in vacuolar Ca²⁺ sequestration through ACA4 and increased extrusion of Ca²⁺ into the root apoplast mediated by ACA10, 12, and 13 would contribute to long-distance retranslocation of root Ca²⁺ to growing shoot tissues which typically are the first to show Ca²⁺ deficiency symptoms.

The only Ca²⁺ antiporters that were identified as showing significant changes in transcript level, were CAX3 and a maize CAX2 homologue. Data on CAX2 are contradictory with one study (Maathuis et al., 2003) reporting a down-regulation and the Genevestigator database depicting up-regulation. Yeast complementation studies suggest that CAX3 may not function as a Ca²⁺ exchanger in planta (Cheng et al., 2003) and expression of CAX2 in tobacco suggests that CAX2 is capable of carrying a range of metals including Ca²⁺, Mn²⁺, and Cd²⁺. Up-regulation of these mechanisms might therefore promote vacuolar Ca²⁺ sequestration or, alternatively, impact on general metal homeostasis including that of micronutrients and heavy metals.

The role of cation exchangers:
The CHX gene family encodes proteins that catalyse transmembrane movement of monovalent cations and are fuelled by the proton motive force. In a few cases a more detailed characterization is available; for example for CHX17 (Cellier et al., 2004). For the most transcriptionally-affected CHX genes (CHX10, 11, 12, 15), expression data (Genevestigator; Cellier et al., 2004) suggest that their expression is extremely low and almost exclusively in pollen. By contrast, CHX17 is significantly expressed in leaves and particularly in root tissue where its transcription was found to be increased by a factor 8 to 9 (Table 2). CHX17 expression levels also rose in response to ABA (Cellier et al., 2004) and chx17 loss of function mutants showed decreased K⁺ content in root tissues. However, no significant differences were observed in Na⁺ content or growth during salinity stress between wild-type and chx17 mutant plants. Collectively, these results suggest that CHX17 transports K⁺ rather than Na⁺ and may be involved in root K⁺ homeostasis particularly during NaCl stress (Cellier et al., 2004).

Non-selective cation channels:
Entry of Na⁺ and other ions into the plant symplast can be beneficial during salinity stress since it will help to decrease the osmotic potential of tissues and hence alleviate water stress. At the same time, however, excessive accumulation of cytoplasmic Na⁺ is highly toxic and the generally prevailing electrochemical Na⁺ gradient would lead to certain cell death if Na⁺ influx were unrestricted. Apart from active Na⁺ extrusion, it is therefore likely that plants at least regulate and probably actively limit Na⁺ influx through potential uptake pathways. In the past decade, great progress has been made in identifying the molecular mechanisms that underlie such pathways, particularly at the functional level through the application of electrophysiological approaches (Tyerman et al., 1997; Maathuis and Sanders, 2001; Demidchik et al., 2002). Such studies have identified non-selective cation channels as the prime suspects that mediate Na⁺ influx. Unfortunately, molecular identification is still largely absent, which greatly hampers in-depth structure–function studies of these particular channels and prevents genetic manipulation. Thus, transcriptomics studies are potentially of great importance to give initial clues to the identity of Na⁺-mediating non-selective channels. Table 2 shows that members of both the glutamate receptor family and the cyclic nucleotide regulated channels are affected at the transcript level by salt stress. Up-regulation and down-regulation occur within both gene families and, as for CNGC19, in a tissue-dependent manner. GLR2.3 was identified in several studies as being down-regulated by salt stress, possibly indicating that it may function in Na⁺ uptake. However, its transcript abundance is also extensively diminished during Ca²⁺ deficiency.

No functional data are available for GLRs, partly since heterologous expression consistently fails to yield functional channels (Davenport, 2002), but it has been suggested that they may play a role in Ca²⁺ transport and Ca²⁺ nutrition. Indeed, GLR3.2 was shown to impact on Ca²⁺ redistribution (Kim et al., 2001) but, significantly, its overexpression also created hypersensitivity to K⁺ and Na⁺. Thus, GLR activity during salt stress may impact on Na⁺ fluxes per se but also on Ca²⁺ and K⁺ nutrition. In contrast to GLR2.3, GLR2.5 is up-regulated in root tissue which may point to a function in the (re)distribution of ions such as K⁺ and Ca²⁺.

For several members of the CNGC family, functional data are available (Leng et al., 2002; Balague et al., 2003) showing that all characterized CNGCs are capable of conducting K⁺ and, apart from CNGC2, Na⁺. As for GLRs, it has been postulated that CNGCs also conduct Ca²⁺. Figure 2 shows more detailed transcriptomics data for the five CNGC isoforms identified as significantly regulated at the transcript level by salinity. In root tissue, transcripts increased up to 9, 27, and 3 times for CNGC1, CNGC19, and CNGC20, respectively. The significance of this up-regulation concerning Na⁺ fluxes, particularly in root tissue, remains to be revealed. The requirement for rapid osmotic adjustment could justify up-regulation of non-selective pathways. By contrast, the CNGC19 transcript was reduced in shoot tissue after salt stress, as was observed for the CNGC3 and CNGC8 transcripts.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Transcript abundance ratios for four CNGCs (cyclic nucleotide gated channels) after salt stress (80 mM NaCl). RNA sampling occurred after 2, 5, 10, 24, and 96 h for shoot and root tissue. The colour bar provides a calibration for the observed ratios; n.d.: not determined.

 
The relevance of the CNGC isoforms with regard to salinity stress was further investigated by comparing growth rates and Na⁺ accumulation between wild type and loss of function mutants. Table 3 shows that growth rates in control conditions and on medium supplemented with 100 mM NaCl are similar for mutants and wild-type seedlings, apart from cngc1 where gene knockout led to improved salt tolerance. Although no significant differences in growth rate were found for the other mutants, measurements of the Na⁺ content in root and shoot tissue after 48 h exposure to 80 mM NaCl showed that, in the cngc3 genotype, shoot [Na⁺] is lower compared with the wild type.

View this table: [in this window] [in a new window]   Table 3. Relative growth rate (RGR) and tissue Na+ content for wild type and CNGC loss of function mutants

 
Taking transcriptomics data as a starting point, the further investigation into the role of the identified CNGCs in salinity stress shows that, for at least one CNGC isoform, CNGC1, a clear effect on salt tolerance can be demonstrated. CNGC1 is predominantly expressed in shoot tissue and is therefore unlikely to impact on Na⁺ influx directly, which might explain the lack of significantly different Na⁺ contents between wild type and knockout mutants. The role of CNGC3 may be more subtle and, since its expression in root tissue is extensive, may impact on uptake, cellular distribution, and long-distance translocation of cations such as Na⁺ and K⁺. In spite of CNGC19 transcript levels increasing almost 30 times during salinity stress, a loss of function of CNGC19 had no obvious effect on salt tolerance as was the case for CNGC20. This suggests that CNGC19 and CNGC20 are ‘false positives’.

In addition to the false positives CNGC19 and CNGC20, GUS reporter studies (results not shown), MPSS data, and microarray data all show that CNGC8 expression is almost exclusively in the pollen, and microarray signals derived for CNGC8 are therefore most likely unreliable. Since homology within the CNGC family is high, cross hybridization could have generated these spurious results.

Putative transporters involved in salinity stress
Table 1 lists approximately 45 transcripts with unknown function that fulfil the criteria outlined earlier. Although all listed transcripts contain four or more transmembrane spans, not all necessarily encode transporters. For example, At4g29330 shows close homology to Der-like (degradation in ER) proteins. Furthermore, some of the gene products that are likely to mediate transport, may not transport cations: the down-regulated transcript At2g02340 shows homology to PHO1, a Pi transporter involved in xylem loading. For other unknown transcripts no mRNA or ESTs are available (Table 1) and thus their expression may be absent or extremely low and array signals may be erroneous. In addition, several of the unknown transcripts also respond to stresses that are not related to salinity. Removal of these ‘false positives’ leaves around 15 transcripts encoding expressed proteins. One of these (At2g17010) contains a mechano-sensitive channel-like domain and, since such channels are generally non-selective, its down-regulation may contribute to limitation of Na⁺ influx. Amongst the up-regulated transcripts are three members of the MATE (proton coupled, multi-antimicrobial extrusion) family (At1g61890, At1g12950, and At2g04050) that show similarity to ‘ripening regulated’ proteins from tomato; genes that are predominantly expressed in fruit tissue and have diverse functions. Thus, apart from the putative mechano-sensitive channel, the relevance of the transcripts listed in Table 1 regarding salinity tolerance remains unknown. The substantial transcriptional regulation of a number of unknown genes may indicate that these do play a significant role in the response to salt/drought stress, but confirmation of this notion will require further approaches such as reverse genetics, overexpression studies, and functional characterization.

Evaluation of transcriptomics approaches
Protein function must respond to internal and external stimuli. Consequently, the activity of proteins is affected by a range of modulatory processes such as protein–protein interactions and phosphorylation, processes, which can cause rapid alterations in protein function. More sustained adaptation may require the adjustment of gene transcript levels as an alternative or in addition to post translational modification. Thus, an assessment of transcript levels may provide insights into the function of specific proteins in particular physiological conditions.

Such an approach may present various pitfalls. For example, data presented in this study suggest that a relatively small number of cation transporters (40) is regulated in response to salt/drought stress and may, therefore, be relevant in plant salinity tolerance. However, a remarkably small proportion of these (around 10% of the total complement of regulated transcripts) emerges from more than one study as being significantly regulated by salinity. This may be partly due to differing conditions and sampling times, but does suggest that transcriptomics analyses per se suffer from a high level of inherent variability.

Second, large numbers of false positives may be generated. For example, the loss of function in transporter genes such as CNGC19, SKOR, and CHX17 had no significant impact on plant salt tolerance in spite of large changes in transcript level during salt stress (Table 2). It should be mentioned, however, that the potential redundancy within transporter families may render a ‘knockout’ strategy unreliable. Conversely, for NHX1 and NHX7 links between transport activity and salt sensitivity are well documented, but with the criteria applied in this study these would not have been identified. The latter shows that transcriptomics studies are likely to generate many false negatives that may include important transcripts. Nevertheless, many of these potential drawbacks can be rectified by complementing transcriptomics approaches with others such as forward and reverse genetics, proteomics, and metabolomics.

The general advantages of transcriptomics studies have been amply documented (Maathuis and Amtmann, 2004). With respect to the impact of salt stress on cation homeostasis many questions remain to be answered regarding transporter regulation, uptake and efflux pathways, and long-distance translocation, and analyses across large numbers of experiments as described here may offer insights and working hypotheses. The large number of identified K⁺ and Ca²⁺ transporters reconfirms the well-documented link between salinity and Ca²⁺ and K⁺ nutrition, and appears to provide a general validation of transcriptomics methodology.

In addition, specific genes were identified that are likely to be important; particularly those that show large changes in transcript level, are identified in independent studies, and show changes specifically to salt stress. However, transcriptomics approaches are likely to be most valuable in uncovering broad associations between particular treatments and physiological processes. Three such correlations can be inferred from this analysis: Apart from reiterating the association between salinity and K⁺ nutrition, data for SKOR and AKT2,3 (Table 2) suggest that increased circulation of K⁺ through the xylem and phloem is an important attribute in maintaining and restoring K⁺ homeostasis during salinity stress. Second, transcriptional regulation of Ca²⁺ transporters in root tissue suggests that salinity induces an effective depletion of Ca²⁺ stores in this organ to augment Ca²⁺ translocation to shoot tissues. Third, transcript levels of many transition metal transporters were found to be regulated, often specifically in response to salt stress. These data point to a hitherto unknown potential link between salinity and the homeostasis of essential transition metals such as Zn²⁺, Cu²⁺, and Fe²⁺. Furthermore, cytoplasmic toxification by transition metals and heavy metals may form an important aspect of salinity stress, which has not received much attention.

	Concluding remarks

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
Transcriptomics studies have greatly contributed to the current quest for functional interpretation of genome sequences. Such studies have helped with gene identification and annotation, explained mutant phenotypes, and delivered many insights into gene function. However, there are several major caveats that limit the contribution of transcriptomics results. First, a phenotype is predominantly determined by the concerted action of proteins and transcriptional regulation does not necessarily correspond with protein activity. Second, transcriptomics methods do not provide a direct link to protein location, which can confound the interpretation of expression studies. These, and the potential pitfalls described above, make transcriptomics studies in isolation of limited value. However, in conjunction with complementary techniques such as reverse and forward genetics or proteomics, it provides a very powerful source to identify important gene candidates, to generate research hypotheses, and to reveal new insights into physiological processes such as salt stress in plants.

	References

Top Abstract Introduction Materials and methods Results and discussion Concluding remarks References

 
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