Most ET estimation using UAVs is based on satellite remote sensing methods. One source energy balance , high resolution mapping of evapotranspiration, machine learning, artificial neural networks, two source energy balance , dual-temperature-difference, the surface energy balance algorithm for Land , and mapping evapotranspiration at high resolution with internalized calibration are introduced in this section. The discussed ET estimation methods with UAVs and their advantages and disadvantages are summarized in Table 3. As mentioned earlier, this article is not intended to provide an exhaustive review of all direct or indirect methods that have been developed for ET estimation, but rather to provide an overview on ET estimation with UAVs applications. Therefore, only those methods which have already been used with the UAVs platform are discussed. Machine learning techniques and ANN models have already been used for estimating hydrological parameters and ecological variables. Due to the ML’s ability to capture non-linear characteristics,microgreen fodder system many research results suggest that machine learning methods can provide better ET estimates than empirical equations based on different meteorological parameters .
Therefore, artificial neural networks were used in to improve the estimation of spatial variability of vine water status. The resulting emissivities were incorporated into the TSEB model to analyze their effects on the estimation of instantaneous energy balance components against ground measurements. Soil salinization causes a significant reduction in the growth and productivity of glycophytes, including major crops. In general, soil salinity is widespread in arid and semi-arid regions, particularly on irrigated land in such areas. However, saline soil is also a serious problem in humid regions such as South and Southeast Asia, where encroachment of sea water occurs through estuaries and groundwater, especially in coastal regions. Approximately 7 % of the total land surface suffers soil salinity to a greater or lesser extent. More than 650 million hectares of land in Asia and Australia are estimated to be salt-affected, which is a serious threat to stable crop production in these densely populated areas.Excessive salt accumulation triggers various detrimental effects due to two major problems: osmotic stress and ion toxicity. Increases in osmotic pressure, caused by salt over-accumulation in the root zone, lead to a reduction in water uptake, which in turn slows down cell expansion and growth, thereby reducing cellular activity. Na+ is a major toxic cation in salt-affected soil environments.
Over-accumulated Na+ outside and inside of plants disturbs K+ homeostasis and vital metabolic reactions, such as photosynthesis, and causes the accumulation of reactive oxygen species. The high-affinity K+ transporter family in plants has been extensively studied since the discovery of the TaHKT2;1 gene from bread wheat , which encodes a Na+ -K+ co-transporter. Analysis of the structure and transport properties of HKT transporters from various plant species has classified these transporter proteins into at least two subfamilies. Class I HKT transporters were found to form a major subfamily that in general exhibits Na+ -selective transport with poor K + permeability. The single HKT1 gene in Arabidopsis thaliana, AtHKT1;1, was found to be essential to cope with salinity stress. Na+ channel activity mediated by AtHKT1;1 was proposed to predominantly function in xylem unloading of Na+ in vascular tissues, particularly in roots, which prevents Na+ over-accumulation in leaf blades in salt stress conditions . In monocot crops such as rice, wheat and barley, HKT genes were found to form a gene family composed of genes encoding class I and class II transporters. QTL analyses for salt tolerance in rice plants detected a strong locus controlling K+ and Na+ contents in shoots, which was subsequently found to encode the OsHKT1;5 transporter. In bread wheat, the Kna1 locus contributing to enhanced K+ -Na+ discrimination in shoots of salt-stressed plants has long been known [25, 26]. In addition, two important independent loci for salt tolerance were also identified in durum wheat.
These were shown to be responsible for maintaining low Na+ concentrations in leaf blades by restricting Na+ transport from roots to shoots. It seems that the Nax2 and Kna1 loci are orthologs, which turned out to encode HKT1;5 transporters. HKT1;5 transporters from rice and wheat plants were demonstrated to mediate Na+ selective transport and maintain a high K/Na ratio in leaf blades during salinity stress by preventing Na+ loading into xylem vessels in the roots, similar to AtHKT1;1. The Nax1 locus has been shown to function in the exclusion of Na+ from leaf sheaths to blades in addition to restricting the movement of Na+ from roots to shoots. Sequencing analysis of the approximate mapping region of the Nax1 locus has suggested that the effect is attributable to the HKT1;4 gene, TmHKT1;4-A2. In rice, a copy of the OsHKT1;4 gene was found in the genome. Recent analysis of the OsHKT1;4 gene of a japonica cultivar and salt-tolerant varieties of indica rice suggested that the level of the OsHKT1;4 transcript correctly spliced in leaf sheaths is closely related to the efficiency of Na+ exclusion from leaf blades upon salinity stress. Furthermore, recent electrophysiological analyses of two TdHKT1;4 transporters from a salt-tolerant durum wheat cultivar reported Na + -selective transport mechanisms with distinct functional features of each transporter. However, ion transport features and the physiological role of OsHKT1;4 in rice remain largely unknown. In this study, we investigated the features of ion transport mediated by OsHKT1;4 using heterologous expression systems. We also characterized the physiological function of OsHKT1;4 under salt stress by analyzing RNAi transgenic rice lines. We found that OsHKT1;4 is a plasma-membrane -localized transporter for mediating selective Na+ transport, and it plays an important role in restricting Na+ accumulation in aerial parts, in particular, in leaf blades during salinity stress at the reproductive growth stage.To investigate the Na+ transport properties of OsHKT1;4, the full length OsHKT1;4 cDNA was isolated from seedlings of the japonica rice cultivar Nipponbare using a specific primer set . The isolated cDNA was 1545 bp long and deduced to encode 500 amino acids, which were completely identical to sequences registered in GenBank. Heterologous expression analysis was performed using a salt hypersensitive mutant of S. cerevisiae . Transgenic G19 cells harboring an OsHKT1;4 expression construct grew with no serious inhibition on arginine phosphate medium in the absence of excess Na+ although the overall growth of OsHKT1;4-expressing cells were slightly weaker than that of cells harboring empty vector .
The addition of 50 mM NaCl triggered severe growth inhibition of OsHKT1;4-expressing cells in contrast to control cells on AP medium . OsHKT1;4-expressing cells accumulated significantly higher levels of Na+ than control cells when cultured in synthetic complete medium containing approximately 2 mM Na+ . Incubation with liquid SC medium supplemented with 25 mM NaCl further stimulated the phenotype,barley fodder system and a significant increase in Na+ accumulation occurred in OsHKT1;4-expTo determine the localization of the OsHKT1;4 protein in plant cells, we fused EGFP at the N-terminus end of OsHKT1;4 and placed under the control of the CaMV35S promoter. Rice protoplasts transformed with EGFP-OsHKT1;4 showed the presence of EGFP fluorescence at the periphery of the cell . Red fluorescence from co-expressed CBL1n-OFP , a PM marker [36], overlapped well with the green fluorescence from EGFP-OsHKT1;4 . In comparison, rice protoplasts co-transformedwith free EGFP and PM-marked CBL1n-OFP showed typical cytoplasmic localization of EGFP , which did not overlap with CBL1n-OFP fluorescence . These results strongly indicated that EGFP-OsHKT1;4 localizes to the PM of rice protoplasts. However, by repeating the transformation experiments several times, we often observed that EGFP-OsHKT1;4 was also present inside the cells and clustered in punctate-like structures . In order to understand if the internal EGFP signal was due to the accumulation of OsHKT1;4 in the secretion pathway, we co-transformed rice protoplasts with EGFPOsHKT1;4 together with an endoplasmic reticulum marker, ER-mCherry. As shown in Fig. 4i-k, EGFPOsHKT1;4 was present in the ER , but was also detectable at the PM , which was not labeled with mCherry . This latter result indicated that EGFP-OsHKT1;4 was partially retained in the ER, but that it was also able to properly reach the PM. Moreover, co-expression of EGFP-OsHKT1;4 with the ER marker revealed that the observed EGFP punctate-like structures were not made of ER membranes, because they did not exhibit mCherry fluorescence. We further investigated if such punctate-like structures could be a part of the Golgi apparatus by co-expressing EGFP-OsHKT1;4 with a Golgi marker, Golgi-mCherry and analyzing optical sections of transformed protoplasts in which the GA was clearly detectable . As shown in Fig. 4o, EGFP and mCherry fluorescence only partially overlapped , with some pwere labeled with EGFP alone . This latter result indicated that EGFP-OsHKT1;4 was also present in the GA as well as in still unidentified structures.We investigated the tissue-specific expression pattern of OsHKT1;4 at various growth stages of rice plants using the same samples reported previously. Higher expression of OsHKT1;4 in leaf sheaths was found throughout the growth periods . At the flowering stage, the highest expression level was found in the peduncle and internode II .
Note that lower levels of OsHKT1;4 expression were also detected in other organs . We further investigated the response of OsHKT1;4 to stress at two different growth stages. At the vegetative growth stage, exposure to 50 mM NaCl resulted in significant reductions in the accumulation of OsHKT1;4 transcripts in all organs except the youngest leaf sheath . A stepwise 25 mM increase in the NaCl concentration every 3 days from 75 mM to 100 mM was subsequently applied to 50 mM NaCl-treated plants, and the same organs were harvested at each NaCl concentration. In general, prolonged and increased NaCl stress maintained severe reductions of OsHKT1;4 expression in young leaf blades, leaf sheaths, basal nodes and roots compared with control plants . One characteristic difference from 50 mM NaCl-treated plants was the expression profile in the youngest leaf sheath , in which OsHKT1;4 expression showed significant reductions as in other tissues, and the decrease-trend became more severe as the strength of NaCl stress increased . At the reproductive stage, OsHKT1;4 transcript levels were significantly increased in peduncles in response to salt stress . In addition, a significant increase in OsHKT1;4 expression was also found in the uppermost node of salt-stressed rice plants compared with control plants, although the basal level of OsHKT1;4 expression in the tissue was relatively low . The node is an essential tissue for distributing minerals, and toxic elements, that are transported from the roots. The node includes different types of vascular bundles such as enlarged VBs and diffuse VBs , each of which have distinct functions in the distribution of elements. Given that the level of expression of OsHKT1;4 was elevated in node I in response to salinity stress , we examined the expression pattern of OsHKT1;4 in EVBs and DVBs by combinational analysis of laser micro-dissection and real-time PCR. As shown in Fig. 6c, OsHKT1;4 expression was predominantly detected in DVBs but not EVBs in node I, which was approximately 28-times higher than the expression in the basal stem .To investigate whether OsHKT1;4-mediated Na+ transport contributes to salt tolerance in rice plants, we generated OsHKT1;4 RNAi plants. Two independent transgenic lines, which showed reductions in OsHKT1;4 expression in leaf sheaths during the reproductive growth phase, were selected and used for phenotypic analysis . Growth with 50 mM NaCl in hydroponic culture for more than 2 weeks in Nipponbare and RNAi lines did not cause any difference in visual characteristics . The Na+ concentration of different organs was compared between WT and RNAi plants after the plants were treated with 50 mM NaCl for 3 days. No difference was found in the Na+ concentration of all organs between WT and RNAi lines . Given that OsHKT1;4 expression in the tissues of rice at the vegetative growth stage was down-regulated, but was up-regulated in some tissues at the reproductive growth stage in response to NaCl stress , we then examined the phenotypes of RNAi lines at the reproductive growth stage in high-salinity conditions. Wild-type Nipponbare plants and each OsHKT1;4 RNAi line were planted in the same pot filled with soil from paddy fields and grown in two independent greenhouse facilities at two different institutes. Nipponbare and OsHKT1;4 RNAi plants were watered with tap water containing 25 mM NaCl when they started heading, and the NaCl concentration was gradually elevated with a 25 mM increase to the maximum concentration of 100 mM for more than a month. Flag leaves, peduncles,nodes and internode IIs were harvested and ion contents were determined.