1. Introduction
Apple,
Malus domestica Borkh, is the commercially grown fruit crop across globe and third-largest produced fruit in India. The cultivation of this crop has seen a drift from commercial cultivation to high-density plantation in recent decades. The orchardists across globe are shifting to high-density plantations due to high returns and better quality produce. However, these high-density orchards require better cultural practices and are more susceptible to water stress conditions. Such orchards cannot be irrigated with conventional flood irrigation and require scheduled irrigation through the drip. Furthermore, the current situation of water scarcity requires more efficient use of land and water resources for horticulture growth as estimates International Water Management Institute in their report, that 25 percent of the world population would experience severe water scarcity by the year 2025 [
1]. Several research studies conducted worldwide show that irrigation water plays a critical role in the overall growth of crops and shows a significant influence on various tree characteristics like plant growth, rootstock function, and quality and disease-free fruit production [
2,
3]. In the long run, it helps in overall rural development, enhancing farmer’s income through a reduction in the input cost of water, fertilizer, insecticide, herbicide, etc., and increases income through higher yields and better quality fruits [
4]. Irrigation scheduling in high-density apple plantations has been reported to be the most significant factor affecting uptake of mineral nutrients, and thus tree growth [
5], and as such, nutrient deficiency and water stress in these orchards are increasingly challenging their success at large scale. Scheduled irrigation is therefore a useful way to maintain a crop’s physiological state and nutrient balance within the crop [
6]. The researchers report that different irrigation methods had different effects on the uptake of nutrient elements [
7]. Many researchers have, however, reported that any reduction in irrigation amount does not hold any significant influence on the concentration of major nutrients like N, P, and K in leaves [
8,
9]. However, it is also seen that the literature on the influence of various irrigation regimes on leaf nutrients mainly focuses on calcium (Ca) and magnesium (Mg) because the moment of these nutrients in plants is primarily by mass flow [
10] and therefore are most affected by irrigation.
As leaves are the primary source of photosynthesis and other physiological activities, it is important to understand the influence of different levels of irrigation on these conditions. Chlorophyll content in leaves has been considered an important trait for crop production. Likewise, leaf relative water content significantly influences photosynthesis and is the appropriate measure of plant water status. As most of the water is lost by evaporation, stomas hold significant importance for monitoring plant–water balance [
11], and therefore it is important to understand how irrigation levels influence stomatal density and structures in plants.
Potassium (K
+) contributes to many physiological and metabolic activities, like maintenance of cellular osmolarity, neutralization of anions, and control of stomatal opening, etc., [
12]. The deficiency of this nutrient significantly reduces photosynthesis resulting in poor growth and development of crops [
13,
14]. For optimal growth, this nutrient must be effectively absorbed by plants from the soil via roots. K
+ transporter genes like KT/HAK/KUP groups are ubiquitously present in plants, which implies their significant role in plant tolerance under water stress conditions [
15,
16]. However, the molecular basis of how different irrigation levels influence the expression of these genes is largely rare. Understanding the importance of these facts and the necessity to observe critical stages of irrigation depending on its influence on various crop phenological stages, which has not been addressed much in earlier studies, this work was undertaken to study the influence of different levels of irrigation on leaf physiological characteristics, nutrient concentration, and expression of transporter genes and to determine critical stages of irrigation for apple under high density plantation.
4. Discussion
The rate of leaf development plays a significant role in fruit productivity of the crop as absorption of photosynthetically active radiations (PAR) and dry matter accumulation primarily depends on the area of leaf and therefore it is imperative to study the influence of irrigation levels on leaf development. The larger the leaf area, the more the PAR is absorbed by the plant and, thus, more accumulation of dry matter. The leaf area under the present investigation significantly increased with increasing irrigation, which may be attributed to more frequent callus tissue formation on the well-watered plants. Our findings are similar to those reported by Gigova et al. [
25] who advocated that water is the most limiting factor affecting proper leaf area development. Kucukyumuk and Kacal [
26] also documented that different irrigation regimes had a significant influence on the leaf area of apples and recorded the highest leaf area in plants frequently irrigated than those irrigated at longer intervals. Similarly, Yuste et al. [
27] in grapes, Klamkowski and Treder [
28] in strawberry, and Eid et al. [
29] in apricot obtained results of a similar trend.
Chlorophyll content in leaves is an important trait for crop production. The corresponding increase in leaf chlorophyll content in plants irrigated at different levels of irrigation at various crop phenological stages was noted to vary between 10 and 31 percent compared to rainfed conditions. Low chlorophyll content in leaves from plants supplied with no irrigation might be due to inhibition of chlorophyll synthesis or disorganization of chloroplasts in the leaves which resulted from water restriction conditions. It might also be due to the significant decrease in mineral contents of leaves, particularly Mg, as Mg is an important constituent of chlorophyll. In the study, leaves from fully irrigated trees had a higher level of Mg content that might have accounted for the higher accumulation of chlorophyll content in leaves from fully irrigated plants. Similar observations were made by Trigo-Córdoba et al. [
30] who reported low chlorophyll content in grape cultivars under rainfed conditions and 50% ETc irrigation. Javadi et al. [
31] in pear, Haifeng et al. [
32] on citrus, and Gholami et al. [
33] in fig also documented higher chlorophyll content under irrigated conditions compared to no irrigation.
Leaf relative water content that signifies the metabolic activity in tissues [
34] indicating the balance between water absorbed and transpired by leaves, declined significantly due to water restriction conditions under lower levels of ETc and rainfed conditions. This decrease in leaf relative water content could have been due to the unavailability of water in the soil and the leaves could not compensate for water lost through transpiration resulting in low water content [
35]. Lower relative water content in plants irrigated only during the flowering and fruit set stage might be due to the reason that leaves during the early season are thin and transpire more water than later in the season. Romero et al. [
36] in almond, Satisha et al. [
37] in grapes, and Alejandro et al. [
38] in apricot also achieved comparable findings in this concern. Stomatal features are known to affect transpiration and, thus, play a vital role in maintaining the water status of plants. The stomata studied were hypostomatic in apple leaves. Kucukyumuk and Kacal [
26] also found that stomas are on the lower epidermis of the apple leaves. Reduction in stomatal density and size of leaves under rainfed conditions might be a response to reduced water loss and cell division under water stress conditions. Carbon assimilation of plants relies on the absorption of CO
2 by stomata, which partially closes to respond to water deficits. Our findings were again in uniformity with Elias [
39] who recorded stomatal closure at periods of strong evaporative demand, which suggested that stomatal density appeared to be primarily influenced by tree water status during the vegetative period in apples. Kour and Bakshi [
40] observed that stomatal aperture and density in leaves from peach seedlings significantly change in response to changing water conditions. Previous reports by Basiouny [
41] in peach, Mısırlı and Aksoy [
42] in figs, and Klamkowski and Treder [
43] and Kawchaya [
44] in strawberry also illustrated a decrease in stomatal density and size due to water stress.
Plant nutrient uptake and subsequent concentrations in various plant parts are influenced by numerous factors including the water status of soil and plant. Water is essential for nutrient uptake by root interception, mass flow, and diffusion. The possible reason for higher nutrient content in leaves of plants subjected to continuous irrigation with drip method may be due to high soil moisture, resulting in the transfer of mineral nutrients and the mass flow of soil solution powered by water absorption and plant root diffusion. The further movement between the soil particles and the rise in mass flow due to a higher transpiration rate as a result of stomata opening improves the transport of nutrients under high soil moisture. Another potential reason for a higher content of leaf nutrients at 100% ETc irrigation levels may be attributed to an extended, more fibrous, and more productive root system, influenced by comparatively improved moisture and thermal regimes, which increased root growth and thus increased the capacity for higher absorption of the nutrient. Less water availability under water stress conditions generally results in a reduction in total nutrient uptake and subsequently reduces the concentration of mineral nutrients in plants [
45].
Ca and Mg uptake is primarily through mass flow [
10], therefore the plants irrigated at 100% and 75% ETc had the highest Ca and Mg concentrations in their leaves. It is because roots absorb more nutrients, especially calcium and magnesium, from the moisture-rich soil compared to dry soil as a result of more extensive root growth [
46]. Furthermore, transpiration rate has important significance for the transport of nutrients from the soil to the top of the plant, and this rate has particular significance for xylem mobile nutrients such as Ca; the decrease in transpiration rate as a consequence of water stress under rainfed conditions might have decreased the uptake of these mobile nutrients. Kacar and Katkat [
47] also have stated that uptake of nutrients increased with transpiration increasing factors. Thaur et al. [
48] reported that drip irrigation at 100% ETc significantly increased leaf nitrogen (N), phosphorus (P), and potassium (K) in apple trees planted under high density plantation. In general, Shirgure et al. [
49] in acid lime, Chauhan and Chandel [
50] in kiwifruit, Küçükyumuk et al. [
51] in cherry, and Zhong et al. [
52] in apple also documented similar results. The variation in gene expression of potassium transporter gene was found to be due to variation in irrigation levels at various crop phenological stages. Variation in transporter genes was also found in accordance with the change in fruit size. Therefore, the availability of water significantly influences gene expression. Similar kinds of variation in nutrient transporter genes have been reported by Dechorgnat et al. [
53], who found that expression of nutrient transporter genes was regulated by nutrient availability and which in turn depends on the availability of water. Therefore, water availability significantly influences nutrient uptake by regulating transporter genes. Similarly, nitrogen inducible transporter showed a strong expression in guard cells and supports the stomatal function in the presence of available forms of nitrogen [
54]. Song et al. [
55] also found that in response to potassium deficiency expression of the potassium transporter gene,
PpeKUP6 was dramatically reduced in peach leaves.