Phenotypic characterization of Arabidopsis thaliana lines overexpressing AVP1 and MIOX4 in response to abiotic stresses

Abstract

Premise

AVP1 (H⁺‐pyrophosphatase) and MIOX4 (myo‐inositol oxygenase) are genes that, when overexpressed individually, enhance the growth and abiotic stress tolerance of Arabidopsis thaliana plants. We propose that pyramiding AVP1 and MIOX4 genes will further improve stress tolerance under water‐limited and salt‐stress conditions.

Methods

MIOX4 and AVP1 reciprocal crosses were developed and phenomic approaches used to investigate the possible synergy between these genes.

Results

Under normal and stress conditions, the crosses had higher foliar ascorbate content than the wild‐type and parental lines. Under water‐limited conditions, the crosses also displayed an enhanced growth rate and biomass compared with the control. The observed increases in photosystem II efficiency, linear electron flow, and relative chlorophyll content may have contributed to this observed phenotype. Additionally, the crosses retained more water than the controls when subjected to salt stress. Higher seed yields were also observed in the crosses compared with the controls when grown under salt and water‐limitation stresses.

Discussion

Overall, these results suggest the combinatorial effect of overexpressing MIOX4 and AVP1 may be more advantageous than the individual traits for enhancing stress tolerance and seed yields during crop improvement.

A significant challenge in agriculture today is the rapidly growing global demand for food, fuel, and fiber. By 2100, global population estimates range between 9.6 and 12.3 billion people (Food and Agriculture Organization, 2010; Gerland et al., 2014). A conceivable answer to help future food and feed demands includes the genetic modification of crops to improve yield, increase nutritional content, and enhance their ability to flourish under limited inputs. The combined loss of yield due to abiotic and biotic stresses is greater than 50% worldwide, with losses caused by abiotic stresses being significantly higher than those caused by biotic agents (Pandey et al., 2017). Salt and water‐limitation stress are the major abiotic stresses that negatively impact plant growth, development, and crop yields, limiting agricultural productivity (Pandey et al., 2015).

Salt stress is the presence of harmful levels of electrolytic minerals in soil or water, which for plants is soils with ≥4 dS m^–1 conductivity (Munns and Tester, 2008). Salinity stress causes a water deficit in plants and has a negative impact on osmosis, inhibiting primary and specialized metabolisms in the cells. The reduction of water potential inside cells under salt stress is due to increased accumulation of Na⁺ and Cl^–, which also disrupts the K⁺/Na⁺ homeostasis, inactivates enzymes, and inhibits biochemical and physiological processes (Munns and Tester, 2008). Water deficit stress inhibits root and shoot development by reducing cell division and elongation, impacts flowering time and number, and inhibits seed production and development (Boyer, 1982).

During abiotic stress conditions, plants accumulate reactive oxygen species (ROS), which, when present in high concentrations, oxidize the biochemical components of cells, such as proteins, DNA, and lipids (Yu and Chan, 2015). Plants produce antioxidant enzymes (e.g., superoxide dismutase and peroxidase) and non‐enzymatic antioxidants (e.g., glutathione, ascorbate [i.e., l‐ascorbic acid, vitamin C], tocopherols, and phenolic compounds) to maintain ROS homeostasis. The ascorbate–glutathione cycle is the major ROS scavenging pathway present in plants, with the ROS scavenger ascorbate being the most abundant antioxidant present in most cell types and in the apoplast (Wheeler et al., 1998; Foyer and Noctor, 2011; Caverzan et al., 2016). In addition to its function as an antioxidant, ascorbate mediates its effect by functioning as a signaling molecule to induce the stress response (Smirnoff, 2011). Ascorbate functions as an electron donor during photosynthesis and protects photoreceptors from ROS (Ishikawa et al., 2006), while also regulating cell proliferation and serving as a substrate for flavonoid and phytohormone biosynthesis (De Tullio and Arrigoni, 2004). Animals have one route for ascorbate biosynthesis, but plants have four pathways: the d‐mannose/l‐galactose (Wheeler et al., 1998), l‐gulose (Wolucka and Van Montagu, 2003), d‐galacturonate (Agius et al., 2003), and myo‐inositol pathways (Lorence et al., 2004).

A small gene family in the model plant Arabidopsis thaliana (L.) Heynh. encodes the myo‐inositol oxygenase (MIOX) enzyme. The MIOX genes are found on four of the five chromosomes in the A. thaliana genome and are named according to their location MIOX1, MIOX2, MIOX4, and MIOX5 (Lorence et al., 2004). The level of MIOX4 expression in particular is directly related to the ascorbate content, with the highest levels measured in the leaves and flowers of A. thaliana compared with the other tissues (Lorence et al., 2004). MIOX4 is the first enzyme present in the myo‐inositol route of ascorbate biosynthesis, and transgenic plants overexpressing the MIOX4 gene have a higher ascorbate content than the controls (Lorence et al., 2004; Lisko et al., 2013; Yactayo‐Chang et al., 2018). The MIOX4 overexpressing (OE) lines display an increased tolerance to abiotic stresses, including salt, cold, heat, and pyrene, a common environmental pollutant (Lisko et al., 2013; Yactayo‐Chang et al., 2018). The molecular mechanisms underpinning these phenotypic effects in the MIOX4 OE lines were recently published (Nepal et al., 2019). Arabidopsis thaliana vacuolar H⁺‐pyrophosphatase (AVP1) is a membrane‐localized enzyme that acidifies the vacuole to power other ionic pumps (Schilling et al., 2017). Arabidopsis thaliana plants overexpressing the AVP1 gene are more tolerant of salt and water‐limitation stresses than the wild type (WT), potentially because of their increased internal storage of solutes and water retention (Gaxiola et al., 2001). The impacts of overexpressing AVP1 or MIOX4 in various plant species are summarized in Table 1.

1 TableEffect of AVP1 overexpression, MIOX4 overexpression, and AVP1 and MIOX4 co‐overexpression in response to abiotic stresses.

Expression type	Gene transformed	Gene source	Species transformed	Stress tested	Observations	Response to stress	References
Single gene	AVP1	A. thaliana	A. thaliana	Salinity	Growth	Tolerant	Gaxiola et al., 2001
			Water deficit	Survival Viability of seeds	Tolerant
AVP1	A. thaliana	S. lycopersicum	Phosphorous deficiency	Root proliferation	Tolerant	Yang et al., 2007
		O. sativa
AVP1	A. thaliana	L. sativa	Nitrate limitation	Shoot and root biomass	Tolerant	Paez‐Valencia et al., 2013
				Lateral root development
AVP1	A. thaliana	S. lycopersicum	Water deprivation	Root biomass	Tolerant	Park et al., 2005
AVP1	A. thaliana	G. hirsutum	Salinity	Growth	Tolerant	Pasapula et al., 2011
				Photosynthetic rate
				Stomatal conductance
				Transpiration rate
				Fiber yield
			Water deficit	Shoot biomass	Tolerant
				Root biomass
AVP1	A. thaliana	S. lycopersicum	Phosphorous deficiency	Fruit yield	Tolerant	Yang et al., 2014
AVP1	A. thaliana	H. vulgare	Salinity	Shoot biomass	Tolerant	Schilling et al., 2014
				Yield
AVP1	A. thaliana	M. sativa	Salinity	Chlorosis	Tolerant	Bao et al., 2009
				Solute and water retention
				Shoot biomass
			Water deficit	Growth	Tolerant
AVP1	A. thaliana	O. sativa	Salinity	Biomass	Tolerant	Kim et al., 2014
				Ion homeostasis
				Photochemical yield (Fv/Fm)
AVP1	A. thaliana	A. stolonifera	Salinity	Growth	Tolerant	Li et al., 2010
				Relative water content
				Solute retention
				Biomass
AVP1	A. thaliana	N. tabacum	Salinity	Growth	Tolerant	Kumar et al., 2014
				Biomass
AVP1	A. thaliana	S. officinarum	Salinity	Growth	Tolerant	Duan et al., 2007
				Chlorosis
MIOX4	A. thaliana	A. thaliana	Heat	Chlorophyll	Tolerant	Tóth et al., 2011
			Light	Photosystem II efficiency
				Non‐photochemical quenching
				Energy‐ dependent quenching
MIOX4	A. thaliana	A. thaliana	Salinity	Growth	Tolerant	Lisko et al., 2013
			Cold	Biomass
			Heat	Root length
			Pyrene
Co‐expression	AVP1	A. thaliana	S. lycopersicum	Salinity	Chlorosis	Tolerant	Bhaskaran and Savithrama, 2011
Na+/H+ vacuolar antiporter	P. glaucum			Chlorophyll
				Proline content
				Ion retention
AVP1	A. thaliana	G. hirsutum	Salinity	Photosynthetic rate	Tolerant	Shen et al., 2015
NHX1	A. thaliana			Root biomass
			Water deficit	Fiber yield	Tolerant
AVP1	A. thaliana	A. thaliana	Salinity	Shoot biomass	Tolerant	Sun et al., 2018
PP2A‐C5	A. thaliana			Root biomass

Note:

AVP1 = ARABIDOPSIS VACUOLAR PYROPHOSPHATASE; MIOX4 = MYO‐INOSITOL OXYGENASE 4; NHX1 = SODIUM/HYDROGEN EXCHANGER 1; PP2A‐C5 = PROTEIN PHOSPHATASE 2A. A. thaliana = Arabidopsis thaliana; A. stolonifera = Agrotis stolonifera; G. hirsutum = Gossypium hirsutum; H. vulgare = Hordeum vulgare; L. sativa = Lactuca sativa; M. sativa = Medicago sativa; N. tabacum = Nicotiana tabacum; O. sativa = Oryza sativa; P. glaucum = Pennisetum glaucum; S. lycopersicum = Solanum lycopersicum; S. officinarum = Saccharum officinarum.

To date, abiotic stress tolerance in plants has mainly been characterized using the manual phenotyping of traits such as plant area, plant height, seed number, panicle number, leaf number, and fruit color. Although manual techniques are useful to characterize plant phenotypes, multiple limitations exist regarding their use. Manual phenotyping is limited to the resolution of the human eye, is error‐prone, and often requires destructive sampling; however, high‐throughput phenotyping methods have emerged to overcome these limitations (Campbell et al., 2018; Yactayo‐Chang et al., 2018; Acosta‐Gamboa, 2019).

In plant phenotyping, multiple imaging sensors, namely visible (RGB), fluorescence, infrared, and hyperspectral light sensors, are used to non‐invasively capture the quantitative traits of plants. Plant phenotyping depends on the absorbed, reflected, and transmitted light in different wavelengths. Each molecule in a cell has a different absorption spectrum; for example, water absorbs light near the infrared region, whereas chlorophyll absorbs light at the blue and red regions (Fiorani and Schurr, 2013; Li et al., 2014). Modern high‐throughput phenotyping using these light sensors is capable of measuring the shape, size, structure, leaf characteristics, fruit parameters, and photosynthetic traits of plants using images, which helps to reduce the biases between individuals collecting the data and between experimental repeats (Campbell et al., 2018; Yactayo‐Chang et al., 2018; Costa et al., 2019). Visible imaging (400–750 nm) is used in phenotyping to track the growth of plants in controlled settings or in the field, and is also capable of measuring abiotic stress responses in terms of growth. Under long salt stress conditions, leaf senescence—a trait that can be used to measure salt tolerance—can be measured by separating the yellow and green areas of leaf images (Munns and Tester, 2008; Rajendran et al., 2009). In fluorescence imaging, chlorophyll complexes fluoresce due to the emission of blue light by chlorophyll, which is observed as chlorophyll fluorescence. These fluorescence parameters tell us about the metabolic status of plants and can be used to detect early stress responses (Gorbe and Catalyud, 2012). Fluorescence imaging is also used to measure photosynthesis parameters and biotic and abiotic stress responses in plants (Balachandran et al., 1997; Chaerle et al., 2007; Konishi et al., 2009). Near infrared (NIR) imaging enables the water content to be measured, as water present in plant tissues absorbs NIR light, which can be quantified (Berger et al., 2010). NIR imaging is commonly used for the measurement of plant responses to water‐limited and salt stress conditions (Seelig et al., 2008; Jones et al., 2009; Acosta‐Gamboa, 2019).

The expression of MIOX4 or AVP1 enhances the abiotic stress tolerance of transgenic plants. Transcriptomic studies of MIOX4 OE plants revealed that AVP1 expression is independent of MIOX4 expression (Nepal et al., 2019); therefore, we hypothesize that the co‐expression of AVP1 and MIOX4 will further improve abiotic stress tolerance. Here, we use high‐throughput phenotyping approaches and real‐time quantitative PCR (qPCR) to characterize the additive effects of AVP1 and MIOX4 in A. thaliana exposed to water‐limitation and salt stress.

MATERIALS AND METHODS

Plant material and growth conditions

Seeds of the A. thaliana AVP1 OE line (AVP1‐1) were provided by R.G. The A. thaliana MIOX4 OE line was developed as described by Nepal et al. (2019). The seeds were surface‐sterilized and the AVP1 OE, MIOX4 OE, and crosses (described below) were sown on Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) plus kanamycin, while the WT Col‐0 controls were sown on MS medium. The plates were stratified at 4°C for three days, transferred to an environmental control chamber, and incubated at 23°C in 65% humidity and a 14 : 10‐h photoperiod with a 165–210 μmol⋅m⁻²⋅s⁻¹ light intensity. After 13 days (water stress treatment) or 14 days (salt stress treatment), vigorous seedlings were transferred to pots containing ProMix PGX (Premier Horticulture, Québec, Canada), with one teaspoon of Osmocote 15‐9‐12 (Scotts Co., Marysville, Ohio, USA) per gallon of soil. Plants were grown in QuickPot 15 (HerkuPlast, Ering, Germany) trays under the conditions cited above for the high‐throughput phenotyping experiments.

Generation of AVP1 × MIOX4 crosses

The MIOX4 OE and AVP1 OE seedlings were grown in MS media; at 10 to 13 days after planting, they were transferred into soil until flowering. Reciprocal crosses were obtained by crossing MIOX4 OE as the pollen donor and AVP1 OE as the pollen recipient and vice versa. The crosses were grown in a growth chamber and allowed to produce siliques, and the resulting seeds were cleaned and planted in MS media supplemented with 50 mg⋅mL⁻¹ kanamycin. The crosses were grown for six generations until homozygous hybrids were obtained. PCRs targeting AVP1, MIOX4, and the kanamycin resistance genes, as well as ascorbate measurements and germination scores, were used for the selection of crosses in each generation as reported in Nepal et al. (2019).

Ascorbate measurements

Arabidopsis thaliana leaves were collected for ascorbate measurement when the plants were in developmental stage 6.1 for the salt stress experiment, and developmental stage 6.3 for the water‐limitation stress, according to the nomenclature presented by Boyes et al. (2001). The ascorbate measurements were performed using an enzyme‐based spectrophotometric method as described previously (Nepal et al., 2019).

RNA extraction and quantitative PCR

qPCR was used for the gene expression analysis, using previously published reference genes and following the Minimum Information for Publication of Quantitative Real‐Time PCR Experiments (MIQE) guidelines (Czechowski et al., 2005; Bustin et al., 2009). Three biological replicates of foliar samples were collected at developmental stages 6.1 and 6.3 for the plants under the salt stress and water‐limitation stress experiments, respectively. The total RNA extraction, quality analysis, and cDNA synthesis were performed as previously described (Nepal et al., 2019).

The efficiencies of the primers for amplifying the AVP1 and MIOX4 genes were calculated using the MIQE guidelines (Bustin et al., 2009). Briefly, four‐fold dilutions of cDNA were obtained, and qPCR was performed using dilutions as 80 ng, 20 ng, 5 ng, 1.25 ng, 0.3125 ng, 0.078125 ng, and 0.01953125 ng of template. The regression line was plotted as the threshold cycle (Ct) value vs. the logarithmic dilution, and the efficiency was calculated using a qPCR efficiency calculator (Thermo Fisher Scientific, Waltham, Massachusetts, USA).

qPCR was performed using a SsoFast EvaGreen supermix (Bio‐Rad Laboratories, Hercules, California, USA) in a CFX384TM real‐time system (Bio‐Rad Laboratories), following the manufacturer’s instructions. GaLDH, ACTIN2, UBQ10, qEF1‐α, UBC, and 18S rRNA were used as reference genes. AVP1, MIOX4, NAM transcription factor–encoding genes, psbA, and HEAT SHOCK PROTEIN (HSP) 17.4 kDa were evaluated as genes of interest (primers and full gene names are provided in Appendix S1). The reference gene validation and gene expression analysis were performed using qBaseplus version 3.0 software (Hellemans et al., 2007; Biogazelle, Zwijnaarde, Belgium). We identified the constitutively expressed reference genes on the basis of their coefficient of variation (CV < 0.5) and gene stability values (M < 0.5). The gene expression was calculated as normalized relative quantities (NRQ) using the formula NRQ = E_goi^ΔCq,goi /n ⁿ√(∏_iE _ref1^ΔCq,ref1) (Hellemans et al., 2007). Three biological replicates and two technical replicates were used for the qPCR.

Water‐limitation treatments

The dry weight and fully saturated weight of each tray were measured using a balance. Twenty trays were used, five for each treatment; of those, four trays were used for the phenotyping and qPCR analyses, while the remaining tray was used for ascorbate quantification. Five genotypes (AVP1 OE, WT, MIOX4 OE, AVP1 × MIOX4, and MIOX4 × AVP1) were randomized in each tray. One well in each tray was kept free of seedlings to enable the measurement of the soil water potential. The desired water saturation level (85%, 50%, 25%, or 12.5%) was applied by weighing the trays and adding water equally to each plant. An initial soil saturation of 85% was used during transplanting.

Salt stress treatment

The dry weight and weight of a fully saturated tray were obtained with a balance. Five genotypes (WT, MIOX4 OE, AVP1 OE, AVP1 × MIOX4, and MIOX4 × AVP1) were randomized in each tray, with two genotypes per tray. One well in each tray was kept free of seedlings to enable the measurement of the soil water potential. Seven days after transplanting, the seedlings were watered to 100% saturation with salt solution (0 mM, 50 mM, 100 mM, and 150 mM) until the seeds were collected.

Soil water potential measurements

Soil water potential (Ψ MPa) was measured using a soil water potential meter (WP4C; Meter Group, Pullman, Washington, USA). All measurements were conducted weekly at the same time of day, as previously described (Acosta‐Gamboa et al., 2017).

High‐throughput phenotyping

Plant images were acquired using a Scanalyzer (LemnaTec, Aachen, Germany), which uses high‐resolution NIR, visible (i.e., RGB), and fluorescence sensors to precisely capture and measure subtle phenotypes. Images of A. thaliana plants were captured every two days from seedlings to fully mature plants (developmental stage 6.3; Boyes et al., 2001). Image acquisition was performed as described by Acosta‐Gamboa et al. (2017). A total of 4320 images (5 genotypes × 8 biological replicates × 4 treatments × 9 time points × 3 cameras) for each experiment were analyzed using the LemnaGrid software. The image analysis was performed as previously described (Arvidsson et al., 2011; Acosta‐Gamboa et al., 2017). Eight biological replicates were grown for each genotype, and each tray contained two genotypes. The data were analyzed using the tray as a random variable and genotype as a fixed variable, with the data for each day being analyzed separately.

Photosynthetic efficiency

A MultispeQ version 1.0 instrument developed by the Kramer Laboratory (Michigan State University; Kuhlgert et al., 2016) was used to measure the photosynthetic efficiency of the plants. The data were downloaded from the PhotosynQ web portal (https://www.photosynq.org/), as described by Kuhlgert et al. (2016). Photosynthetic efficiency was calculated by measuring the chlorophyll fluorescence under different light conditions, as described by Baker et al. (2007). In addition, the following photosynthesis parameters were determined at developmental stage 6.1 for the salt stress experiments and developmental stage 6.3 for the water‐limitation stress experiments: photosystem II (PSII) efficiency (ΦII), non‐photochemical quenching (NPQt), linear electron flow (LEF), and relative chlorophyll content. Eight biological replicates were measured for each genotype.

Seed yield

Seed number was calculated as described by Acosta‐Gamboa et al. (2017).

Statistical analysis

One‐way ANOVAs in combination with Tukey’s post‐hoc tests were used to determine the statistical significance of the differences between genotypes at the P < 0.05 level.

RESULTS

Selection of homozygous AVP1 and MIOX4 crosses

We developed AVP1 and MIOX4 reciprocal crosses, which were grown for six generations to obtain homozygous lines. The crosses displaying enhanced growth were selected for the abiotic stress experiments. These lines were found to have a higher ascorbate content than the AVP1 OE line and the WT (Fig. 1A). We confirmed that the AVP1 and MIOX4 genes of interest were significantly expressed in the crosses (Fig. 1B, C). The lines with the highest ascorbate contents and highest AVP1 and MIOX4 expression levels were used for the phenotypic characterization under salt and water‐limitation stresses.

1 Figure. Selection of homozygous AVP1 OE and MIOX4 OE crosses for the abiotic stress study. (A) Ascorbate content of the AVP1 × MIOX4 and MIOX4 × AVP1 crosses, the wild type, and the parent lines at developmental stage 5.0 (Boyes et al., 2001). Data are means ± SE (n = 6). Different letters indicate statistically significant differences at P < 0.05, as determined using a one‐way ANOVA and Tukey’s post‐hoc test. (B) MIOX4 and (C) AVP1 expression in the studied genotypes, relative to the levels of UBQ10 and 18S rRNA expression. Data are means ± SE of three replicates for each genotype. Different letters indicate statistically significant differences at P < 0.05, as determined using a one‐way ANOVA and Tukey’s post‐hoc test. FW, fresh weight; WT, wild type; MIOX4, MIOX4 OE line; AVP1‐1, AVP1 OE line; AXM, AVP1 × MIOX4 cross; MXA, MIOX4 × AVP1 cross.

Soil water potential decreased when plants were exposed to abiotic stress

The next step was to verify the application of salt stress and water‐limitation stress to plants using water potential as an indicator. We found that 26 days after germination, there was a significant difference in the soil water potential among the different water saturation treatments used (85%, 50%, 25%, and 12.5% of the fully saturated condition) (Appendix S2A). Similarly, under salt stress, we found a significant difference in the soil water potential among the treatments 28 days after germination (Appendix S2B). Under salt stress, the WT and MIOX4 OE lines were more stressed than AVP1 OE and the AVP1 × MIOX4 crosses (Fig. 2A, B). Similarly, under water limitation, the AVP1 × MIOX4 crosses were less stressed than the WT and parent lines (Fig. 2A, C). These results indicate that the salt and water‐limitation stresses were successfully applied to the treatment groups and that plant responses to the abiotic stresses were detectable.

2 Figure. Representative images of plants subjected to abiotic stress taken using the visible light sensor. (A) Plants under normal growth conditions (soil at 85% of full saturation with water). (B) Plants under the salt‐stress condition (150 mM NaCl). (C) Plants under water‐limitation stress (12.5% of full saturation). WT, wild type; MIOX4, MIOX4 OE line; AVP1‐1, AVP1 OE line; AXM, AVP1 × MIOX4 cross; MXA, MIOX4 × AVP1 cross.

Ascorbate was elevated in the AVP1 × MIOX4 crosses grown under abiotic stress

Ascorbate is a multifunctional molecule found in plants and a potent ROS scavenger. We therefore analyzed the expression of AVP1 and MIOX4, and the abundance of ascorbate in the crosses under water‐limitation and salt stress. We analyzed the expression of AVP1 and MIOX4 in the water‐limitation experiment, using GaLDH, UBC, and EF1‐α as reference genes. The expression of AVP1 was significantly elevated in AVP1 OE and the crosses compared with the MIOX4 OE and WT plants under both 85% water saturation (control) and water‐limited (12.5% saturation) conditions (Fig. 3A). MIOX4 was highly expressed in MIOX4 OE and the crosses grown under control and water‐limited conditions (Fig. 3B). We found that, under 85% water saturation, the foliar ascorbate content of the selected crosses was significantly higher than that of AVP1 OE and the WT, and was comparable to the MIOX4 OE line. Under 12.5% water saturation, the ascorbate content was significantly higher in the crosses and the MIOX4 OE compared with AVP1 OE and the WT (Fig. 3C). Elevated ascorbate contents were also detected in the AVP1 × MIOX4 crosses growing under 50% and 25% water saturation (Appendix S3A, B).

3 Figure. Expression analysis of AVP1 and MIOX4 and the ascorbate content in the AVP1 × MIOX4 crosses subjected to abiotic stress. (A) MIOX4 expression at 85% water saturation (control) and water‐limitation stress (12.5% saturation). (B) AVP1 expression under water‐limitation stress. (C) Ascorbate content in plants grown under water‐limitation stress for one week. (D) AVP1 expression in the control (0 mM NaCl) and salt‐stress (150 mM NaCl) treatments. (E) MIOX4 expression under salt stress. (F) Ascorbate content in plants grown under salt stress for two weeks. Different letters indicate statistically significant differences at P < 0.05, as determined using a one‐way ANOVA and Tukey’s post‐hoc test. Data are means ± SE (n = 3). The foliar tissues were obtained at developmental stage 6.3 for the water‐limitation experiments and at developmental stage 6.1 for the salt‐stress experiments (Boyes et al., 2001). WT, wild type; MIOX4, MIOX4 OE line; AVP1‐1, AVP1 OE line; AXM, AVP1 × MIOX4 cross; MXA, MIOX4 × AVP1 cross.

We analyzed the expression of AVP1 and MIOX4 in the plants exposed to salt stress, using GaLDH, UBC, and ACTIN2 as the reference genes. AVP1 was significantly more highly expressed in AVP1 OE and the crosses compared with MIOX4 OE and the WT under both the control and salt‐stress (150 mM NaCl) treatments (Fig. 3D). Similarly, MIOX4 was much more highly expressed in MIOX4 OE and the crosses than in AVP1 OE or the WT, both in the control and salt‐stress conditions (Fig. 3E). Similarly, the ascorbate content was significantly higher in the crosses and MIOX4 OE than in AVP1 OE or the WT under both the control and salt‐stress conditions (Fig. 3F). Furthermore, ascorbate was also higher in the crosses than the other genotypes when grown under 50 mM or 100 mM NaCl (Appendix S3C, D). These results confirmed that the AVP1 × MIOX4 crosses had elevated expression levels of AVP1 and MIOX4 under normal, water‐limited, and salt‐stress conditions, with elevated ascorbate contents under both abiotic stresses. This elevated MIOX4 expression and corresponding increase in ascorbate content in the MIOX4 OE line and AVP1 × MIOX4 crosses might help plants maintain ROS homeostasis under abiotic stress conditions. Additionally, the overexpression of AVP1 in AVP1 OE and AVP1 × MIOX4 might help plants to sequester solutes into the vacuole and protect them from ionic toxicity.

Expression levels of master regulator genes were elevated in the AVP1 × MIOX4 crosses grown under stress conditions

A transcriptomics study of the MIOX4 OE line revealed its increased expression of abiotic stress tolerance genes (Nepal et al., 2019). Here, we performed an expression analysis of the heat‐ and salt‐tolerance genes under water‐limitation and salt‐stress treatments. We found that the expression of a heat shock protein (HSP 17.4 kDa) was significantly lower in the WT grown under 85% water saturation (Fig. 4A) and 12.5% water saturation (Fig. 4B) than the transgenic lines. For the salt‐stress experiments, we explored the expression of the NAM transcription factor genes, revealing they were expressed at significantly lower levels in the WT and AVP1 OE plants than in the AVP1 × MIOX4 crosses and MIOX4 OE (Fig. 4C) in the absence of salt stress. In plants grown under 150 mM salt stress, the expression levels of the NAM transcription factor genes were elevated compared with those of the controls. Additionally, under 150 mM salt stress, the expression of the NAM transcription factor gene was significantly higher in MIOX4 OE and the crosses than in the WT and AVP1 OE lines (Fig. 4D). These results indicate that WT plants were more susceptible to water limitation and salt stress than the transgenic lines. Furthermore, the AVP1 × MIOX4 crosses were transcriptionally primed for salt stress tolerance.

4 Figure. Expression analysis of HSP 17.4 kDa and the NAM transcription factor genes under abiotic stress. (A, B) Expression of HSP 17.4 kDa gene in (A) 85% water saturation (control condition) and (B) water‐limitation stress (12.5% water saturation) relative to the expression levels of GaLDH, UBC, and EF1‐α. (C, D) Expression of the NAM transcription factor genes in (C) control (0 mM NaCl) and (D) salt‐stress conditions (150 mM NaCl) relative to the expression levels of GaLDH, UBC, and ACTIN2. Different letters indicate statistically significant differences at P < 0.05, as determined using a one‐way ANOVA and Tukey’s post‐hoc test. Data are means ± SE (n = 3). The foliar tissues were obtained at developmental stage 6.3 for the water‐limitation experiments and at developmental stage 6.1 for the salt‐stress experiments (Boyes et al., 2001). WT, wild type; MIOX4, MIOX4 OE line; AVP1‐1, AVP1 OE line; AXM, AVP1 × MIOX4 cross; MXA, MIOX4 × AVP1 cross.

The AVP1 × MIOX4 crosses had enhanced biomass under abiotic stress

We leveraged a high‐throughput platform to analyze the biomass of AVP1 × MIOX4 crosses under abiotic stress conditions. The projected leaf surface area of all genotypes of interest was measured throughout the vegetative life cycle. The growth rate was relatively higher in AVP1 OE and the AVP1 × MIOX4 crosses than in MIOX4 OE and the WT grown under 85% water saturation (Fig. 5A). The growth rate of AVP1 × MIOX4 (A×M) crosses was the highest under the water‐limitation stress (Fig. 5B), while the growth rate of AVP1 × MIOX4 (M×A) crosses was significantly higher than MIOX4 OE and the WT, but similar to AVP1 OE, under the water‐limited condition (Fig. 5B). These results indicate that one of the AVP1 × MIOX4 crosses had a higher biomass under water‐limitation stress than the parent lines.

5 Figure. The AVP1 × MIOX4 crosses accumulate enhanced biomass under water‐limitation stress. (A, B) Projected leaf areas of plants under (A) 85% water saturation (control) and (B) water‐limitation stress (12.5% water saturation). (C, D) Projected leaf areas of plants under (C) control (0 mM NaCl) and (D) salt stress (150 mM NaCl). Data are means ± SE (n = 8). Different letters in the box indicate statistically significant differences (P < 0.05) on that specific day, as determined using a one‐way ANOVA and Tukey’s post‐hoc test. WT, wild type; MIOX4, MIOX4 OE line; AVP1‐1, AVP1 OE line; AXM, AVP1 × MIOX4 cross; MXA, MIOX4 × AVP1 cross.

Furthermore, in the absence of salt stress, the projected leaf surface areas of the crosses were higher than that of MIOX4 OE and the WT (Fig. 5C). Additionally, under 150 mM salt stress, the projected leaf surface area of the crosses was higher than for MIOX4 OE and the WT (Fig. 5D). Under salt stress, the AVP1 × MIOX4 crosses have an enhanced biomass compared with the MIOX4 OE line and the WT, and a comparable biomass to the AVP1 OE line.

Taken together, these results indicate that the AVP1 × MIOX4 (A×M) cross has an enhanced biomass compared with the parent lines and WT under water‐limitation stress and a biomass comparable to AVP1, while the AVP1 × MIOX4 (M×A) cross is more successful under water‐limitation stress. The differences observed in biomass under water limitation in the reciprocal crosses might be due to “parent‐of‐origin” effects. The reciprocal crosses may differ in the growth factors and nutrients present in the maternal plant, the inheritance of maternal mitochondria, and differences in epigenetic imprinting between plants (Fosella, 2001).

The AVP1 × MIOX4 crosses had enhanced PSII efficiency under abiotic stress

The conversion of light, water, and carbon dioxide into carbohydrates is critical for the productivity of plants. We therefore measured PSII efficiency using a MultispeQ, revealing that under both well‐watered (85% water saturation) and water‐limited conditions (12.5% water saturation), PSII efficiency was higher in the AVP1 × MIOX4 crosses than in MIOX4 OE or the WT (Fig. 6A). Under 0 mM NaCl, the PSII efficiency was higher in the crosses than in MIOX4 OE and the WT. Under the 150 mM salt treatment, the PSII efficiency was relatively higher in the AVP1 × MIOX4 (A×M) lines than the other genotypes (Fig. 6D).

6 Figure. Photosynthetic efficiency is increased in the AVP1 × MIOX4 crosses grown under abiotic stress. (A) The photosystem II (PSII) efficiency of plants under 85% saturation (control) and water‐limitation stress (12.5% water saturation). (B, C) The PSII reaction center gene psbA (AtCg0020) expression levels under the (B) control and (C) water‐limitation stress conditions (n = 3). (D) PSII efficiency of plants grown under control (0 mM NaCl) and salt‐stress (150 mM NaCl) conditions (n = 8). (E, F) psbA expression in plants under (E) control and (F) salt‐stress conditions (n = 3). Data are means ± SE. Different letters indicate differences at 0.05 significance level. Different letters indicate statistically significant differences at P < 0.05, as determined using a one‐way ANOVA and Tukey’s post‐hoc test. WT, wild type; MIOX4, MIOX4 OE line; AVP1‐1, AVP1 OE line; AXM, AVP1 × MIOX4 cross; MXA, MIOX4 × AVP1 cross.

We analyzed the expression of the PSII center related gene psbA (AtCg00020), which was previously found to be upregulated in MIOX4 OE in normal, water‐limited, and salt‐stress conditions (Nepal et al., 2019). We found that psbA expression was significantly lower in the WT compared with MIOX4 OE, AVP1 OE, and the AVP1 × MIOX4 crosses grown under both 85% (Fig. 6B) and 12.5% water saturation (Fig. 6C). Similarly, psbA expression was significantly lower in the WT than in the transgenic plants under 0 mM NaCl (Fig. 6E) and 150 mM NaCl (Fig. 6F). These results indicate that PSII efficiency was significantly increased in the AVP1 × MIOX4 crosses under water‐limitation stress, and that the WT was more susceptible to water‐limitation and salt stresses.

Chlorophyll content was higher and chlorosis was lower in the AVP1 × MIOX4 crosses under abiotic stress

We measured the relative chlorophyll content of all genotypes using a MultispeQ and examined chlorosis using the visible sensor of our phenomics platform. We found that the chlorophyll content of the crosses was higher than that of MIOX4 OE and the WT under 85% and 12.5% water saturation (Fig. 7A). The relative chlorophyll content was also significantly higher in the AVP1 × MIOX4 crosses under 0 mM and 150 mM NaCl (Fig. 7C) compared with MIOX4 OE and the WT. Under 12.5% water saturation, the AVP1 × MIOX4 crosses exhibited less chlorosis than the parent lines and the WT (Fig. 7B). Similarly, under 150 mM salt stress, chlorosis was lower in the crosses than in MIOX4 OE and the WT (Fig. 7D). These results indicate that the chlorophyll content of the AVP1 × MIOX4 lines was more stable than that of the parent lines and the WT under water‐limitation stress.

7 Figure. Relative chlorophyll content is increased in the AVP1 × MIOX4 crosses. (A) The chlorophyll contents of plants grown under control (85% water saturation) and water‐limitation stress (12.5% water saturation) conditions. (B) Chlorosis in plants under water‐limitation stress. (C) The chlorophyll contents of plants grown under control (0 mM NaCl) and salt‐stress (150 mM NaCl) conditions. (D) Chlorosis in plants under salt stress. Yellow color indicates chlorosis detected with the RGB sensor. Data are means ± SE (n = 8). Different letters indicate statistically significant differences at P < 0.05, as determined using a one‐way ANOVA and Tukey’s post‐hoc test. WT, wild type; MIOX4, MIOX4 OE line; AVP1‐1, AVP1 OE line; AXM, AVP1 × MIOX4 cross; MXA, MIOX4 × AVP1 cross.

AVP1 × MIOX4 crosses grown under abiotic stress exhibit higher LEF and lower NPQt

We found that, under 85% water saturation (Fig. 8A) and without salt stress (Fig. 8C), the LEF was similar in all genotypes at developmental stages 6.3 and 6.1, respectively. Under the water‐limitation (12.5% water saturation) stress, the LEF was higher in the AVP1 × MIOX4 crosses compared with the parent lines and the WT (Fig. 8A). Similarly, under 150 mM salt stress, the LEF was significantly higher in the crosses compared with MIOX4 OE and the WT (Fig. 8C). These results indicate that the AVP1 × MIOX4 crosses had enhanced LEF compared with the parent lines and the WT grown under abiotic stress.

8 Figure. Linear electron flow (LEF) is increased and non‐photochemical quenching (NPQt) is decreased in the AVP1 × MIOX4 crosses under abiotic stress. (A) The LEF in plants under control (85% water saturation) and water‐limitation stress (12.5% water saturation) conditions. (B) The NPQt values of plants under control and water‐limitation stress conditions. (C) The LEF in plants grown under control (0 mM NaCl) and salt stress (150 mM NaCl) conditions. (D) The NPQt values of plants grown under control and salt‐stress conditions. Data are means ± SE (n = 8). Different letters indicate statistically significant differences at P < 0.05, as determined using a one‐way ANOVA and Tukey’s post‐hoc test. WT, wild type; MIOX4, MIOX4 OE line; AVP1‐1, AVP1 OE line; AXM, AVP1 × MIOX4 cross; MXA, MIOX4 × AVP1 cross.

The NPQt values obtained from the MultispeQ were measured in plants grown under abiotic stresses and control conditions. The NPQt values of the AVP1 × MIOX4 crosses were lower than in MIOX4 OE and the WT under 85% water saturation. Under 12.5% water saturation, the NPQt values of the crosses were lower than the parent lines and the WT (Fig. 8B). In the absence of salt stress, the NPQt values were significantly lower in the AVP1 × MIOX4 crosses than in MIOX4 OE and the WT. Similarly, in plants exposed to 150 mM salt stress, the NPQt values of the AVP1 × MIOX4 lines were lower than MIOX4 OE and the WT (Fig. 8D). These results indicate that the AVP1 × MIOX4 crosses dissipated less light as heat compared with the parent lines and the WT under water‐limitation stress, and less than MIOX4 OE and the WT under salt stress.

Water retention was higher in the AVP1 × MIOX4 crosses grown under salt stress

Water retention is important for enabling plants to cope with abiotic stress and maintain normal physiological processes. We found that the crosses retained relatively more water than MIOX4 OE and WT under 85% water saturation (Appendix S4A). In contrast, the level of water retention was similar in all genotypes under the 12.5% water‐limitation stress (Appendix S4B). In the absence of salt stress, the crosses retained relatively more water than MIOX4 OE and WT (Appendix S4C). Under 150 mM NaCl, the water retention of the crosses was higher than the parent lines and the WT (Appendix S4D). These results indicate that the AVP1 × MIOX4 lines were more efficient at retaining water than MIOX4 OE and the WT under salt and water‐limitation stresses.

AVP1 × MIOX4 crosses displayed lower levels of high‐signal chlorophyll fluorescence than the WT and parent lines under salt stress

We used the fluorescence sensor of our high‐throughput phenotyping platform to measure the effect of stress on the plants. A high in planta chlorophyll fluorescence signal indicates stress and senescence in plants. We found that, under 85% water saturation, the AVP1 × MIOX4 crosses were relatively less stressed than MIOX4 OE and the WT (Appendices S5A, S6A). Similarly, under 12.5% water limitation, the crosses were less stressed than MIOX4 OE and the WT (Appendices S5B, S6C). Both without stress and under 150 mM NaCl, the AVP1 × MIOX4 lines were relatively less stressed than MIOX4 OE and the WT (Appendices S5C, D, S6B). These results indicate that the AVP1 × MIOX4 crosses displayed lower levels of high‐signal chlorophyll fluorescence than their counterparts when challenged by salt and water‐limitation stresses.

The seed yield of the AVP1 × MIOX4 crosses was higher than the WT under stress conditions

Seed numbers were manually counted. We found that under 85% water saturation, the seed yield was higher in AVP1 × MIOX4 crosses than in the WT and comparable with that of the parent lines (Fig. 9A). Relative to the seed number under 85% water saturation, we observed a 50% yield loss across all genotypes when subjected to 50% water saturation (Appendix S7A). In plants grown under 25% water saturation, the AVP1 × MIOX4 crosses produced more seeds than MIOX4 OE and the WT (Appendix S7B). There was a significant penalty in seed production in plants of all genotypes subjected to 12.5% water saturation relative to the yields under 85% water saturation; under this water‐limitation stress, the AVP1 × MIOX4 (A×M) cross produced significantly more seeds than the parent lines and the WT (Fig. 9A). Despite this, the decrease in seed production between the 85% and 12.5% water saturation conditions was relatively higher in the AVP1 × MIOX4 crosses compared with the parent lines and the WT.

The AVP1 × MIOX4 crosses produced higher seed yields when grown under abiotic stresses. (A) Seed yields of plants grown under control (85% water saturation) and water‐limitation stress (12.5% water saturation) conditions. (B) Seed yields of plants grown under control (0 mM NaCl) and salt‐stress (150 mM NaCl) conditions. Different letters indicate statistically significant differences at P < 0.05, as determined using a one‐way ANOVA and Tukey’s post‐hoc test. Data are means ± SE (n = 8). WT, wild type; MIOX4, MIOX4 OE line; AVP1‐1, AVP1 OE line; AXM, AVP1 × MIOX4 cross; MXA, MIOX4 × AVP1 cross.

In the absence of salt stress, seed yields were significantly lower in the WT than in the parent lines and the AVP1 × MIOX4 crosses (Fig. 9B). Under 50 mM salt stress, no significant difference in yield was observed between the genotypes (Appendix S7C). In contrast, under 100 mM NaCl salt stress, we measured a 54.65% yield reduction in the WT, a 32.75% reduction in MIOX4 OE, a 5.44% reduction in AVP1 OE, a 7.57% reduction in the AVP1 × MIOX4 (A×M) cross, and a 2.09% reduction in the AVP1 × MIOX4 (M×A) cross compared with their yields under the 0 mM salt treatment (Appendix S7D). Similarly, under 150 mM NaCl, the seed yields were reduced by 67% in the WT, 48.4% in MIOX4 OE, 18.63% in AVP1 OE, 6.04% in the AVP1 × MIOX4 (A×M) cross, and 7.75% in the AVP1 × MIOX4 (M×A) cross compared with the control. The seed yields of the AVP1 × MIOX4 crosses were significantly higher than those of MIOX4 OE and the WT (Fig. 9B). These results indicate that seed production in the AVP1 × MIOX4 crosses was less impacted by salt stress than it was in the parent lines and the WT.

DISCUSSION

We previously reported that the overexpression of MIOX4 increased the ascorbate content, enhanced the biomass, and improved the abiotic stress tolerance of the resulting plants (Lorence et al., 2004; Lisko et al., 2013). Ascorbate is an abundant antioxidant molecule in plants that maintains ROS homeostasis. A transcriptomic analysis of MIOX4 OE has shown that the expression of abiotic stress tolerance genes confers tolerance against abiotic stress (Nepal et al., 2019). Similarly, the overexpression of AVP1 leads to abiotic stress tolerance in different plant species (Gaxiola et al., 2001; Schilling et al., 2014). AVP1 expression is independent of MIOX4 expression in A. thaliana (Nepal et al., 2019); therefore, the constitutive expression of both AVP1 and MIOX4 in a single plant was expected to further improve their abiotic stress tolerance. This was demonstrated here using high‐throughput phenotyping to capture the physiology and development of these plants under stress conditions. Visible sensors were also used here to measure abiotic stress tolerance by tracking growth parameters, such as as leaf morphology and leaf chlorosis (Munns and Tester, 2008; Rajendran et al., 2009). In A. thaliana, projected leaf surface area and chlorosis have previously been used to measure plant tolerance of water‐limitation stress and salt stress (Acosta‐Gamboa, 2019).

The overexpression of either MIOX4 or AVP1 confers tolerance of multiple abiotic stresses, such as salinity, water deficit, heat, light, and environmental pollutants (Lisko et al., 2013; Schilling et al., 2017; Yactayo‐Chang, 2018; Acosta‐Gamboa, 2019). Transgenic plants overexpressing either of these genes have a higher ascorbate content, which is known to enhance tolerance against salt stress (Liu et al., 2015; Zhang et al., 2015; Lim et al., 2016; Yactayo‐Chang et al., 2018) and water‐limitation stress (Eltayeb et al., 2006). Here, we showed that the ascorbate content of AVP1 OE was relatively higher than the WT (Fig. 1A). The AVP1 enzyme uses energy obtained from the hydrolysis of inorganic pyrophosphate (PPi) to orthophosphate (Pi), which is increased in AVP1 OE (Gaxiola et al., 2001). The GDP‐galactose phosphorylase enzyme uses Pi to convert GDP‐d‐mannose into GDP‐l‐galactose, an important step in ascorbate biosynthesis (Wheeler et al., 1998; Schilling et al., 2017). In this study, we developed reciprocal AVP1 × MIOX4 crosses with a high ascorbate content, in addition to elevated AVP1 and MIOX4 expression levels (Fig. 3). These crosses had higher growth rates under normal and salt stress conditions (Fig. 5 A–D). The enhanced growth results we observed were consistent with results reported by other teams working with other species (Gaxiola et al., 2001; Park et al., 2005; Pasapula et al., 2011; Lisko et al., 2013).

Under the water‐limitation stress, the AVP1 × MIOX4 (A×M) cross displayed enhanced growth relative to the WT and parent lines (Fig. 5B). This may be due to the pleiotropic effects caused by expressing MIOX4 and AVP1. MIOX4 OE plants were previously reported to have higher biomass, which was attributed to their increased ascorbate levels, as well as more intracellular auxin biosynthesis, auxin transport, cell elongation, and an increased photosynthetic efficiency (Nepal et al., 2019). The overexpression of AVP1 enhances growth and biomass accumulation, as well as increasing CO₂ fixation, sugar oxidation, ATP biosynthesis, and photosynthesis (Khadilkar et al., 2016). Furthermore, AVP1 helps in auxin polar transport and the relocation of sucrose to sink tissues (Schilling et al., 2017). The enhanced growth of the AVP1 × MIOX4 crosses under water‐limitation conditions is therefore likely due to the additive effects of overexpressing AVP1 and MIOX4.

Under the normal, water‐limited, and salt‐stress conditions, ascorbate was higher in the AVP1 × MIOX4 crosses than in AVP1 OE and the WT (Fig. 3C, F). During the water‐limitation stress, ascorbate was elevated in all genotypes compared with the level under normal conditions (Fig. 3C). Ascorbate acts as an antioxidant and scavenges ROS produced during photorespiration and under abiotic stress conditions (Noctor and Foyer, 1998; Foyer and Noctor, 2011). We found that the NAM transcription factor genes were significantly upregulated in MIOX4 OE and the crosses under normal and salt stress conditions (Fig. 4C, D). This is consistent with a recent finding that high‐ascorbate plants had elevated NAM transcription factor gene expression levels under normal conditions (Nepal et al., 2019). The overexpression of the NAM transcription factor genes was previously shown to increase the tolerance of rice (Oryza sativa L.) against water‐limitation and salt stress via their transactivating activity (Hu et al., 2016). Increased ascorbate levels in the AVP1 × MIOX4 lines might help them maintain ROS homeostasis and protect plants from oxidative damage to cellular components under abiotic stress conditions.

The photosynthetic efficiency of the parent lines and AVP1 × MIOX4 crosses was relatively higher than the WT under normal (Fig. 6A, D), water‐limited (Fig. 6A), and salt‐stress conditions (Fig. 6D). Furthermore, psbA was significantly overexpressed in the parent lines and crosses compared with the WT under normal, water‐limited, and salt‐stress conditions (Fig. 6B–F). The constitutive expression of AVP1 enhances photosynthetic efficiency and tolerance of salt stress (Pasapula et al., 2011; Kim et al., 2013). Similarly, cotton (Gossypium hirsutum L.) plants transgenically expressing A. thaliana AVP1 and NHX1 (SODIUM/HYDROGEN EXCHANGER 1) are tolerant of salinity stress and exhibit increased photosynthetic efficiency (Shen et al., 2015). The NHX1 Na⁺/H⁺ antiporter actively exports Na⁺ ions outside the cells (Munns and Tester, 2008). Plants constitutively overexpressing MIOX4 were previously shown to be tolerant of heat and light stress, with enhanced PSII efficiency (Tóth et al., 2011). The AVP1 × MIOX4 crosses are tolerant of salt and water‐limited stress, likely due to their higher photosynthetic efficiency.

The light absorbed by chlorophyll pigments is either dissipated as heat, NPQt, or fluorescence, and electrons are transferred from PSII to PSI to produce ATP. Fluorescence imaging can be used to measure chlorophyll fluorescence and thereby measure early stress responses in plants (Gorbe and Catalyud, 2012). In A. thaliana, chlorophyll fluorescence has been used to measure plant tolerance of water‐limitation and salt stress (Acosta‐Gamboa, 2019). Here, we found that the relative chlorophyll content was higher in the AVP1 × MIOX4 (A×M) cross under 85% water saturation, and higher in the AVP1 × MIOX4 (M×A) cross than MIOX4 OE and the WT under the water‐limited condition (Fig. 7A). Chlorosis was reduced in AVP1 OE and the AVP1 × MIOX4 crosses compared with MIOX4 OE and the WT under water‐limitation conditions (Fig. 7B). Under the salt stress condition, the relative chlorophyll content is higher in AVP1 OE and the AVP1 × MIOX4 crosses than in MIOX4 OE and the WT, and chlorosis is reduced in these lines (Fig. 7C, D). Salt stress, light stress, and heat stress–tolerant plants were shown to have a high chlorophyll content and reduced chlorosis (Duan et al., 2007; Bao et al., 2009; Tóth et al., 2011). Similarly, LEF was significantly higher in the crosses under a salt stress treatment (Fig. 8C) and relatively higher under the water‐limitation stress (Fig. 8A). Salt stress–tolerant AVP1 OE cotton and rice have enhanced photosynthesis (Kim et al., 2013; Shen et al., 2015). Furthermore, NPQt was lower in the crosses under normal, water‐limited, and salt‐stress conditions (Fig. 8B, D). Non‐photochemical quenching was also low in heat and light stress–tolerant MIOX4 OE (Tóth et al., 2011). Increased chlorophyll content, decreased chlorosis, and lower NPQt may help the AVP1 × MIOX4 crosses to tolerate salt and water‐limitation stresses.

Near‐infrared sensor imaging was used to measure the relative water content of plants under abiotic stress (Seelig et al., 2008; Jones et al., 2009; Acosta‐Gamboa, 2019). Relative water retention was higher in the AVP1 × MIOX4 crosses than the other genotypes under normal conditions and the salt‐stress treatment (Appendix S4C, D). Alfalfa (Medicago sativa L.) and creeping bent grass (Agrostis stolonifera L.) transgenically expressing A. thaliana AVP1 both had increased water retention and were tolerant of salt stress (Bao et al., 2009; Li et al., 2010). The AVP1 × MIOX4 crosses were less stressed than the WT and parent lines during the normal, water‐limited, and salt‐stress treatments (Appendix S5). Under salt‐stress conditions, the relative water retention of the AVP1 × MIOX4 crosses was higher, which helped plants to tolerate salt stress.

The seed yields of the AVP1 × MIOX4 crosses, MIOX4 OE, and AVP1 OE were higher than the WT under normal and salt‐stress conditions (Fig. 9A, B). Previously, the transgenic expression of AVP1 was found to increase fruit yields in tomato (Solanum lycopersicum L.) under phosphorous deficiency (Yang et al., 2014). Similarly, the yields of transgenic wheat (Triticum aestivum L.) expression AVP1 were higher than the controls under salinity stress (Schilling et al., 2014). Under the water‐limitation stress, the seed yields were significantly higher in the AVP1 × MIOX4 crosses (Fig. 9B). The co‐expression of NHX1 and AVP1 increased fiber yields in cotton (Shen et al., 2015). The overexpression of MIOX4 increased seed yields under water‐limitation stress, salinity stress, and heat stress (Acosta‐Gamboa, 2019). The increased yield pf the AVP1 × MIOX4 crosses can be attributed to the additive effects of the enhanced expression of AVP1 and MIOX4.

Plants overexpressing individual AVP1 and MIOX4 genes are tolerant of multiple abiotic stresses. High‐throughput phenotyping is powerful approach to measure the tolerance of plants to abiotic stresses in a non‐destructive fashion. Visible sensors have made it possible to quantify the projected leaf surface area and chlorosis to measure stress tolerance simultaneously in a large number of samples. This study shows that the simultaneous expression of AVP1 and MIOX4 further improves tolerance to water‐limitation stress, while the salt stress tolerance of the AVP1 × MIOX4 crosses relative to MIOX4 OE and the WT was comparable to that of AVP1 OE. The AVP1 × MIOX4 crosses had increased LEF and enhanced photosynthetic efficiency. Additionally, the crosses utilized light energy more efficiently, dissipating less heat. The AVP1 × MIOX4 crosses produced more seeds under the water‐limitation stress, which, alongside their decreased chlorosis, reflected their improved water‐limitation stress tolerance. It would be interesting to test the effect of pyramiding the AVP1 and MIOX genes in A. thaliana using hyperspectral imaging due to the ability of that sensor to provide information about the chemical composition of plant tissues. In crop plants of economic importance, these genes could be introduced as a strategy for improving their nutritional content and resilience to abiotic stresses, as evaluated using high‐throughput phenotyping and hyperspectral imaging.

Acknowledgments

The authors thank S. S. Cunningham, A. Villalpa‐Arroyo, A. Wilkie, and K. Lee for technical support; and M. Dolan, F. Goggin, T. Marsico, F. Medina‐Bolivar, K. Medina‐Jimenez, and Z. Campbell for providing helpful suggestions. This work was supported by the National Science Foundation (NSF award numbers IIA‐1430427 and 1736192 to A.L.). The Scanalyzer was acquired with funds from the Plant Powered Production Center (NSF grant EPS‐0701890). We also thank the Arkansas Biosciences Institute and the Arkansas Research Alliance for additional financial support. N.N. thanks the Molecular Biosciences Ph.D. program at Arkansas State University for partial stipend support.

AUTHOR CONTRIBUTIONS

N.N. performed the experiments, prepared figures, and wrote the initial draft of the manuscript; J.P.Y.C., R.G., A.W., and J.M. generated the AVP1 and MIOX4 crosses; C.L.A. helped to conduct the abiotic stress experiments; and R.G. and A.L. initially conceived the experiments and wrote the final draft of the manuscript.