ROS was suggested to involve in cell elongation through an effect on cell-wall loosening [20–22], participation in signaling  and induction of degradation of macromolecules such as chlorophyll, membrane lipids, proteins and RNA under stress conditions or senescence [23, 24]. Under salinity, reduction in ROS content was reported as one of the factors involved in leaf growth restriction [7, 9], but ROS were also reported to increase under salinity  and to involve in salinity-induced leaf damages . Consequently, ROS is considered today to have a dual effect under salinity, on one hand they are required for normal growth of young cells, but on the other they can induce damage especially in mature tissue. In the present study (Figure 4), in accord with previous studies [7, 9], salinity was observed to reduce ROS levels in the growing cells. Various ROS scavengers are active in the plant cells, such as ascorbate peroxidase (APX), superoxide dismutases (SODs), glutathione S-transferase, and are known to involve in ROS detoxification during salt stress [2, 7, 23]. In the growing cells from the leaf base, APX and SOD activity was demonstrated to be significantly higher under NaCl . The aim of the current study was to identify genes that are affected by salinity specifically at defined locations along the growing zone (i.e., correlate to defined stages of tissue expansion in the leaf) and may therefore involve in leaf growth restriction at defined stages of cell development. Due to the central roles suggested for ROS in facilitating cell growth and induction of cellular damage, it is not surprising that changes in ROS contents were identified in the growing cells, and that a considerable proportion (about 13%) of the affected genes belonged to the antioxidant group (Figure 2). Furthermore, the difference in sensitivity to salinity between cells from the two developmental stages evaluated, was reflected also in differences in antioxidant related proteins in the transcriptome.
Numerous antioxidant genes were found to be induced by salinity in the present study and most of these were isolated from the 15–30 mm region that was characterized by highest growth. At this region cell elongation is maximal (Figure 1),  and consequently there is a high demand for ROS for cell loosening . The observed induction of genes involved in antioxidant defense at this early growth stage, resulted in lower ROS levels and consequently growth restriction observed previously by Neves-Piestun and Bernstein  and Rodriquez et al., . For example, Isovaleryl-CoA dehydrogenase (IVDH), a key enzyme in the ETF/ETFQO complex in the mitochondria that induce an alternative respiration pathway that prevents ROS production in mitochondria under various conditions  was up-regulated by NaCl in the young tissue segment. ETF/ETFQO complex was reported to be induced under oxidative stress  and during dark induced senescence , and IVDH was shown to be essential for its activity . Transaldolase, a key enzyme in the pentose phosphate pathway, which is the main NADPH-producing pathway. NADPH is involved in both the thioredoxin redox cycle and the glutathione redox cycle against ROS [30, 31]. Additionally, expression of jasmonate-responsive (JR) genes was found to involve in resistance to oxidative stress . 3-isopropylmalate dehydratase, which catalyzes the second step in the biosynthesis of leucine and is involved in the methionine chain elongation cycle for glucosinolate formation , was identified as a JR gene and involved in oxidative response . The induced carbonic anhydrase was previously reported to be elevated during pathogen attack and consequently high ROS activity , and to act as an antioxidant and active player in plant disease resistance . The induced oxidoreductase acyl-[acyl-carrier-protein] desaturase can have an antioxidant effect as well, because it can reduce O- to two molecules of water . The overexpression of this enzyme in tobacco under chilling stress significantly reduced ROS levels in the plant tissue and allowed elevated activity of antioxidant enzymes . A phosphoserine phosphatase that is involved in amino acid production  can have oxidative effect as well since amino acids are known as antioxidants in plants  and phosphoserine phosphatase was reported previously to involve in stress tolerance . Aspartate aminotransferase as well can be involved in antioxidant defense by the production of glutamate. It was previously reported that the glutamate metabolic flux was dominant in oxidative defense under water stress . The acyl-CoA-binding protein was found to induce plant tolerance to various stresses including oxidative stress [40, 41].
Additional genes can be involved in the antioxidant defense due to their activity in metabolic pathways. An overexpression of dihydroflavonol-4-reductase, which is involved in anticyonine production, led to cell-death resistance in rice due to reduction of hydrogen peroxide concentration . Moreover the elevated NADPH levels observed in a rice mutant  can be supplied to the glutathione redox cycle for protection against ROS . Surprisingly, important ROS scavengers such as superoxide dismutases (SODs), catalases (CATs), peroxidases and ascorbate peroxidase (APX), were not affected on the transcriptome level in the studied regions of the growth zone. Expression analysis for APX by Real-Time PCR supported this result (Figure 3). This is also in accord with a previous study from our lab which identified highest activity level of APX at the basal 15 mm region from the leaf base .
In light of the high expression of genes involved in antioxidant defense under salinity, it is suggested that they are the cause of the low ROS levels in the elongation region under salinity, and consequently the reduced cell-wall loosening. The plants have a very effective antioxidant system that facilitates maintenance of a tight ROS balance in the cell , and easily adapts to changes in ROS under abiotic stresses . Indeed, in the distal part of the growth zone, only two genes involved in antioxidant activity were identified, and one of them was reduced under stress. This could result from the well documented reduction in photosynthetic activity under salinity . Thereby, the plant adaptive strategy to prevent excess of ROS under stress condition by a massive expression of ROS scavengers, results in a negative effect on growth, i.e., growth restriction. From a plant survival, and thereby evolutionary perspective, growth restriction should be less damaging than the direct tissue damage which could occur by the stress-induced elevation of ROS.
In addition to genes belonging to the antioxidant defense system, various genes that can be classified into energy, photosynthetic and structural groups were affected as well by salinity. The elevation of these genes reflects the earlier maturation of the salt-stressed leaf tissues compared to the control; represent involvement in protection against the NaCl damages by an antioxidative defense; or participation in cell growth restriction mechanisms. For instance, reduced levels of ACC oxidase in salt-stressed plants in the 30–50 mm region that points to higher ethylene production , likely correspond to the difference in tissue age between the two treatments. I.e., the tissue found at the distal region of growth zone in salt-stressed plants is older than in the control treatment. This correlates with the observed elevation under salinity of the hydroxyproline-rich glycoprotein (HRGP), that is a component of the cell-wall produced in maize in an ethylene-dependent process, in the same region . This elevation suggests an increase in cell-wall rigidity since HRGPs contain short rigid blocks of contiguous O-glycosylated hydroxyproline residues that involve in intermolecular cross-linking and cell-wall formation . The dirigent protein pDIR9 was elevated as well under stress. Dirigent proteins are involved in lignin biosynthesis, act as physical barriers, have a strengthening role and repair damaged cell-walls . Consequently they can increase rigidity under stress due to increased lignification and reduced cell-wall loosening. Effect of NaCl on these three proteins therefore points to earlier maturation of cells under salt stress and higher rigidity of the cell-wall under stress. The resulted higher rigidity under salinity will also reduce cell-wall loosening and hence the ability for cell enlargement. Elevation of these proteins under salinity in cells of the 30–50 mm region therefore correlates with the heightened growth sensitivity to salinity of this region compared to the younger region found 15–30 mm from the base (Tables 1 and 2). Earlier cessation of growth under salinity was demonstrated for the leaves studied in the present project (Figure 1) as well as in several other studies with monocot leaves [4, 9, 10].
Some of the genes identified by the SSH, such as the elongation factor EF1A or cyclophyllin, are known to have chaperones activity [48, 49]. Salinity is known to promote protein dysfunction and reduce protein stability, and chaperones and heat-shock proteins are considered to involve in salt resistance by sustaining protein stability and function and prevention of protein aggregation . In the young cells from the 15–30 mm region, three different Hsp70 were induced (Table 3), while in the 30–50 mm zone no genes that their products have chaperone functions were isolated and moreover, two isolated EFs were down-regulated pointing at lower protection against stress. Moreover, it was previously suggested that Hsp70 is involved in protein trafficking to peroxisomes  and consequently can affect peroxisome antioxidant activity. Again, these results correlate with the higher sensitivity to salinity of the older cells from the 30–50 mm region compared to the younger tissue from the 15–30 mm region. Overexpression of DnaK1 (a member of the Hsp70 group) in tobacco was demonstrated before to induce salt tolerance . Taken together, these results demonstrate more protection against the stress in the 15–30 mm region compared to the 30–50 mm region, correlating to the difference in the extent of stress-induced growth reduction between these two regions.
Under salinity, increased energy demand for maintenance processes such as compartmentation and osmotic adjustment, coupled with reduced energy production via effects on the photosynthetic apparatus, might reduce energy availability in the plant. Sensitivity of growing cells to salinity might therefore be affected by localized energy-generating biochemical processes and genes involved in energy supply. As an adaptation mechanism, inorganic pyrophosphatase (H+-PPase) activity can be induced. H+PPase, can replace glycolytic ATP consuming enzyme reactions partially by reactions which utilize inorganic pyrophosphate (PPi) as an alternative energy source . Induction of H+-PPase by NaCl increased with distance from the leaf base, i.e., cell age, along the growth zone. The smallest change was observed at the region of highest growth (15–30 mm from the leaf base). The higher expression of PPi at the distal part of the growth zone, where the cells are older, may suggest that the demand for alternative energy increases with prolonged exposure to the stress. At the same time vacuolar H+-PPase was isolated at the younger region. It can supply energy similar to PPi and can supplement energy demands at the region of highest growth. At 15–30 mm from leaf base (the center of the growing zone) additional genes involved in energy metabolism were induced by salinity, and overall this zone was affected by salinity more than the older zone, marking it as the primary affected cell developmental stage.