Effective root responses to salinity stress include maintained cell expansion and carbon allocation.

Acclimation of root growth is vital for plants to survive salt stress. Halophytes are great examples of plants that thrive even under severe salinity, but their salt tolerance mechanisms, especially those mediated by root responses, are still largely unknown. We compared root growth responses of the halophyte Schrenkiella parvula with its glycophytic relative species Arabidopsis thaliana under salt stress and performed transcriptomic analysis of S. parvula roots to identify possible gene regulatory networks underlying their physiological responses. Schrenkiella parvula roots do not avoid salt and experience less growth inhibition under salt stress. Salt-induced abscisic acid levels were higher in S. parvula roots compared with Arabidopsis. Root transcriptomic analysis of S. parvula revealed the induction of sugar transporters and genes regulating cell expansion and suberization under salt stress. 14 C-labeled carbon partitioning analyses showed that S. parvula continued allocating carbon to roots from shoots under salt stress while carbon barely allocated to Arabidopsis roots. Further physiological investigation revealed that S. parvula roots maintained root cell expansion and enhanced suberization under severe salt stress. In summary, roots of S. parvula deploy multiple physiological and developmental adjustments under salt stress to maintain growth, providing new avenues to improve salt tolerance of plants using root-specific strategies.

Arabidopsis Sucrose Synthase 3 (SUS3) regulates starch accumulation in guard cells at the end of day.

Starch in the stomatal guard cells is largely synthesized using carbon precursors originating from sugars imported from the leaf mesophyll. Such heterotrophic nature of guard cell starch synthesis prompted us to investigate the role of cytosolic sucrose synthases (SUS) in this pathway. Out of the six members of the Arabidopsis SUS gene family, SUS3 was the most highly expressed isoform in guard cells. The Arabidopsis sus3 mutant displayed changes in guard cell starch contents comparable to the Wild Type (WT) up until 6 h into the day. After this time point, sus3 guard cells surprisingly started to accumulate starch at very high rates, reaching the end of the day with significantly more starch than WT. Based on the phenotype of the sus3 mutant, we suggest that in guard cells, SUS3 is involved in the regulation of carbon fluxes towards starch synthesis during the second half of the day. SUS3 may be part of a previously predicted guard cell futile cycle of metabolic reactions, in which sucrose is re-synthesized from UDP-glucose to avoid excessive starch synthesis toward the end of the day. This is in contrast to typical storage organs, in which cytosolic SUS is required to produce ADP-glucose for starch synthesis.

Starch biosynthesis in guard cells has features of both autotrophic and heterotrophic tissues.

The pathway of starch synthesis in guard cells (GCs), despite the crucial role starch plays in stomatal movements, is not well understood. Here, we characterized starch dynamics in GCs of Arabidopsis (Arabidopsis thaliana) mutants lacking enzymes of the phosphoglucose isomerase-phosphoglucose mutase-ADP-glucose pyrophosphorylase starch synthesis pathway in leaf mesophyll chloroplasts or sugar transporters at the plastid membrane, such as glucose-6-phosphate/phosphate translocators, which are active in heterotrophic tissues. We demonstrate that GCs have metabolic features of both photoautotrophic and heterotrophic cells. GCs make starch using different carbon precursors depending on the time of day, which can originate both from GC photosynthesis and/or sugars imported from the leaf mesophyll. Furthermore, we unravel the major enzymes involved in GC starch synthesis and demonstrate that they act in a temporal manner according to the fluctuations of stomatal aperture, which is unique for GCs. Our work substantially enhances our knowledge on GC starch metabolism and uncovers targets for manipulating GC starch dynamics to improve stomatal behavior, directly affecting plant productivity.

Arabidopsis guard cell chloroplasts import cytosolic ATP for starch turnover and stomatal opening.

Stomatal opening requires the provision of energy in the form of ATP for proton pumping across the guard cell (GC) plasma membrane and for associated metabolic rearrangements. The source of ATP for GCs is a matter of ongoing debate that is mainly fuelled by controversies around the ability of GC chloroplasts (GCCs) to perform photosynthesis. By imaging compartment-specific fluorescent ATP and NADPH sensor proteins in Arabidopsis, we show that GC photosynthesis is limited and mitochondria are the main source of ATP. Unlike mature mesophyll cell (MC) chloroplasts, which are impermeable to cytosolic ATP, GCCs import cytosolic ATP through NUCLEOTIDE TRANSPORTER (NTT) proteins. GCs from ntt mutants exhibit impaired abilities for starch biosynthesis and stomatal opening. Our work shows that GCs obtain ATP and carbohydrates via different routes from MCs, likely to compensate for the lower chlorophyll contents and limited photosynthesis of GCCs.

Mesophyll-derived sugars are positive regulators of light-driven stomatal opening.

Guard cell membrane ion transport and metabolism are deeply interconnected, and their coordinated regulation is integral to stomatal opening. Whereas ion transport is exceptionally well understood, how guard cell metabolism influences stomatal movements is less well known. Organic metabolites, such as malate and sugars, fulfill several functions in guard cells during stomatal opening as allosteric activators, counter-ions, energy source and osmolytes. However, their origin and exact fate remain debated. Recent work revealed that the guard cell carbon pool regulating stomatal function and plant growth is mostly of mesophyll origin, highlighting a tight correlation between mesophyll and guard cell metabolism. This review discusses latest research into guard cell carbon metabolism and its impact on stomatal function and whole plant physiology.

Glucose uptake to guard cells via STP transporters provides carbon sources for stomatal opening and plant growth.

Guard cells on the leaf epidermis regulate stomatal opening for gas exchange between plants and the atmosphere, allowing a balance between photosynthesis and transpiration. Given that guard cells possess several characteristics of sink tissues, their metabolic activities should largely depend on mesophyll-derived sugars. Early biochemical studies revealed sugar uptake into guard cells. However, the transporters that are involved and their relative contribution to guard cell function are not yet known. Here, we identified the monosaccharide/proton symporters Sugar Transport Protein 1 and 4 (STP1 and STP4) as the major plasma membrane hexose sugar transporters in the guard cells of Arabidopsis thaliana. We show that their combined action is required for glucose import to guard cells, providing carbon sources for starch accumulation and light-induced stomatal opening that are essential for plant growth. These findings highlight mesophyll-derived glucose as an important metabolite connecting stomatal movements with photosynthesis.

Guard Cell Starch Degradation Yields Glucose for Rapid Stomatal Opening in Arabidopsis.

Starch in Arabidopsis (Arabidopsis thaliana) guard cells is rapidly degraded at the start of the day by the glucan hydrolases α-AMYLASE3 (AMY3) and ß-AMYLASE1 (BAM1) to promote stomatal opening. This process is activated via phototropin-mediated blue light signaling downstream of the plasma membrane H+-ATPase. It remains unknown how guard cell starch degradation integrates with light-regulated membrane transport processes in the fine control of stomatal opening kinetics. We report that H+, K+, and Cl- transport across the guard cell plasma membrane is unaltered in the amy3 bam1 mutant, suggesting that starch degradation products do not directly affect the capacity to transport ions. Enzymatic quantification revealed that after 30 min of blue light illumination, amy3 bam1 guard cells had similar malate levels as the wild type, but had dramatically altered sugar homeostasis, with almost undetectable amounts of Glc. Thus, Glc, not malate, is the major starch-derived metabolite in Arabidopsis guard cells. We further show that impaired starch degradation in the amy3 bam1 mutant resulted in an increase in the time constant for opening of 40 min. We conclude that rapid starch degradation at dawn is required to maintain the cytoplasmic sugar pool, clearly needed for fast stomatal opening. The conversion and exchange of metabolites between subcellular compartments therefore coordinates the energetic and metabolic status of the cell with membrane ion transport.

Peeling back the layers of crassulacean acid metabolism: functional differentiation between Kalanchoë fedtschenkoi epidermis and mesophyll proteomes.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that offers the potential to engineer improved water-use efficiency (WUE) and drought resilience in C3 plants while sustaining productivity in the hotter and drier climates that are predicted for much of the world. CAM species show an inverted pattern of stomatal opening and closing across the diel cycle, which conserves water and provides a means of maintaining growth in hot, water-limited environments. Recent genome sequencing of the constitutive model CAM species Kalanchoë fedtschenkoi provides a platform for elucidating the ensemble of proteins that link photosynthetic metabolism with stomatal movement, and that protect CAM plants from harsh environmental conditions. We describe a large-scale proteomics analysis to characterize and compare proteins, as well as diel changes in their abundance in guard cell-enriched epidermis and mesophyll cells from leaves of K. fedtschenkoi. Proteins implicated in processes that encompass respiration, the transport of water and CO2 , stomatal regulation, and CAM biochemistry are highlighted and discussed. Diel rescheduling of guard cell starch turnover in K. fedtschenkoi compared with that observed in Arabidopsis is reported and tissue-specific localization in the epidermis and mesophyll of isozymes implicated in starch and malate turnover are discussed in line with the contrasting roles for these metabolites within the CAM mesophyll and stomatal complex. These data reveal the proteins and the biological processes enriched in each layer and provide key information for studies aiming to adapt plants to hot and dry environments by modifying leaf physiology for improved plant sustainability.

Salinity in Autumn-Winter Season and Fruit Quality of Tomato Landraces.

Tomato landraces, originated by adaptive responses to local habitats, are considered a valuable resource for many traits of agronomic interest, including fruit nutritional quality. Primary and secondary metabolites are essential determinants of fruit organoleptic quality, and some of them, such as carotenoids and phenolics, have been associated with beneficial proprieties for human health. Landraces' fruit taste and flavour are often preferred by consumers compared to the commercial varieties' ones. In an autumn-winter greenhouse hydroponic experiment, the response of three Southern-Italy tomato landraces (Ciettaicale, Linosa and Corleone) and one commercial cultivar (UC-82B) to different concentrations of sodium chloride (0 mM, 60 mM or 120 mM NaCl) were evaluated. At harvest, no losses in marketable yield were noticed in any of the tested genotypes. However, under salt stress, fresh fruit yield as well as fruit calcium concentration were higher affected in the commercial cultivar than in the landraces. Furthermore, UC-82B showed a trend of decreasing lycopene and total antioxidant capacity with increasing salt concentration, whereas no changes in these parameters were observed in the landraces under 60 mM NaCl. Landraces under 120 mM NaCl accumulated more fructose and glucose in the fruits, while salt did not affect hexoses levels in UC-82B. Ultra-performance liquid chromatography-tandem mass spectrometry analysis revealed differential accumulation of glycoalkaloids, phenolic acids, flavonoids and their derivatives in the fruits of all genotypes under stress. Overall, the investigated Italian landraces showed a different behaviour compared to the commercial variety UC-82B under moderate salinity stress, showing a tolerable compromise between yield and quality attributes. Our results point to the feasible use of tomato landraces as a target to select interesting genetic traits to improve fruit quality under stress conditions.

The Thioredoxin-Regulated α-Amylase 3 of Arabidopsis thaliana Is a Target of S-Glutathionylation.

Reactive oxygen species (ROS) are produced in cells as normal cellular metabolic by-products. ROS concentration is normally low, but it increases under stress conditions. To stand ROS exposure, organisms evolved series of responsive mechanisms. One such mechanism is protein S-glutathionylation. S-glutathionylation is a post-translational modification typically occurring in response to oxidative stress, in which a glutathione reacts with cysteinyl residues, protecting them from overoxidation. α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. The Arabidopsis genome contains three genes encoding α-amylases. The sole chloroplastic member, AtAMY3, is involved in osmotic stress response and stomatal opening and is redox-regulated by thioredoxins. Here we show that AtAMY3 activity was sensitive to ROS, such as H2O2. Treatments with H2O2 inhibited enzyme activity and part of the inhibition was irreversible. However, in the presence of glutathione this irreversible inhibition was prevented through S-glutathionylation. The activity of oxidized AtAMY3 was completely restored by simultaneous reduction by both glutaredoxin (specific for the removal of glutathione-mixed disulfide) and thioredoxin (specific for the reduction of protein disulfide), supporting a possible liaison between both redox modifications. By comparing free cysteine residues between reduced and GSSG-treated AtAMY3 and performing oxidation experiments of Cys-to-Ser variants of AtAMY3 using biotin-conjugated GSSG, we could demonstrate that at least three distinct cysteinyl residues can be oxidized/glutathionylated, among those the two previously identified catalytic cysteines, Cys499 and Cys587. Measuring the pK a values of the catalytic cysteines by alkylation at different pHs and enzyme activity measurement (pK a1 = 5.70 ± 0.28; pK a2 = 7.83 ± 0.12) showed the tendency of one of the two catalytic cysteines to deprotonation, even at physiological pHs, supporting its propensity to undergo redox post-translational modifications. Taking into account previous and present findings, a functional model for redox regulation of AtAMY3 is proposed.

The evolution of functional complexity within the ß-amylase gene family in land plants.

BACKGROUND: ß-Amylases (BAMs) are a multigene family of glucan hydrolytic enzymes playing a key role not only for plant biology but also for many industrial applications, such as the malting process in the brewing and distilling industries. BAMs have been extensively studied in Arabidopsis thaliana where they show a surprising level of complexity in terms of specialization within the different isoforms as well as regulatory functions played by at least three catalytically inactive members. Despite the importance of BAMs and the fact that multiple BAM proteins are also present in other angiosperms, little is known about their phylogenetic history or functional relationship. RESULTS: Here, we examined 961 ß-amylase sequences from 136 different algae and land plant species, including 66 sequenced genomes and many transcriptomes. The extraordinary number and the diversity of organisms examined allowed us to reconstruct the main patterns of ß-amylase evolution in land plants. We identified eight distinct clades in angiosperms, which results from extensive gene duplications and sub- or neo-functionalization. We discovered a novel clade of BAM, absent in Arabidopsis, which we called BAM10. BAM10 emerged before the radiation of seed plants and has the feature of an inactive enzyme. Furthermore, we report that BAM4 - an important protein regulating Arabidopsis starch metabolism - is absent in many relevant starch-accumulating crop species, suggesting that starch degradation may be differently regulated between species. CONCLUSIONS: BAM proteins originated sometime more than 400 million years ago and expanded together with the differentiation of plants into organisms of increasing complexity. Our phylogenetic analyses provide essential insights for future functional studies of this important class of storage glucan hydrolases and regulatory proteins.

Quantification of Starch in Guard Cells of Arabidopsis thaliana.

In this protocol, we describe how to quantify starch in guard cells of Arabidopsis thaliana using the fluorophore propidium iodide and confocal laser scanning microscopy. This simple method enables monitoring, with unprecedented resolution, the dynamics of starch in guard cells.

Starch as a determinant of plant fitness under abiotic stress.

Contents 943 I. 943 II. 944 III. 945 IV. 945 V. 948 VI. 949 950 References 950 SUMMARY: Abiotic stresses, such as drought, high salinity and extreme temperatures, pose one of the most important constraints to plant growth and productivity in many regions of the world. A number of investigations have shown that plants, including several important crops, remobilize their starch reserve to release energy, sugars and derived metabolites to help mitigate the stress. This is an essential process for plant fitness with important implications for plant productivity under challenging environmental conditions. In this Tansley insight, we evaluate the current literature on starch metabolism in response to abiotic stresses, and discuss the key enzymes involved and how they are regulated.

High-Throughput Non-destructive Phenotyping of Traits that Contribute to Salinity Tolerance in Arabidopsis thaliana.

Reproducible and efficient high-throughput phenotyping approaches, combined with advances in genome sequencing, are facilitating the discovery of genes affecting plant performance. Salinity tolerance is a desirable trait that can be achieved through breeding, where most have aimed at selecting for plants that perform effective ion exclusion from the shoots. To determine overall plant performance under salt stress, it is helpful to investigate several plant traits collectively in one experimental setup. Hence, we developed a quantitative phenotyping protocol using a high-throughput phenotyping system, with RGB and chlorophyll fluorescence (ChlF) imaging, which captures the growth, morphology, color and photosynthetic performance of Arabidopsis thaliana plants in response to salt stress. We optimized our salt treatment by controlling the soil-water content prior to introducing salt stress. We investigated these traits over time in two accessions in soil at 150, 100, or 50 mM NaCl to find that the plants subjected to 100 mM NaCl showed the most prominent responses in the absence of symptoms of severe stress. In these plants, salt stress induced significant changes in rosette area and morphology, but less prominent changes in rosette coloring and photosystem II efficiency. Clustering of ChlF traits with plant growth of nine accessions maintained at 100 mM NaCl revealed that in the early stage of salt stress, salinity tolerance correlated with non-photochemical quenching processes and during the later stage, plant performance correlated with quantum yield. This integrative approach allows the simultaneous analysis of several phenotypic traits. In combination with various genetic resources, the phenotyping protocol described here is expected to increase our understanding of plant performance and stress responses, ultimately identifying genes that improve plant performance in salt stress conditions.

Starch Turnover and Metabolism during Flower and Early Embryo Development.

The accumulation of starch within photosynthetic tissues and within dedicated storage organs has been characterized extensively in many species, and a function in buffering carbon availability or in fueling later growth phases, respectively, has been proposed. However, developmentally regulated starch turnover within heterotrophic tissues other than dedicated storage organs is poorly characterized, and its function is not well understood. Here, we report on the characterization of starch turnover during flower, early embryo, and silique development in Arabidopsis (Arabidopsis thaliana) using a combined clearing-staining technique on whole-mount tissue. Besides the two previously documented waves of transient starch accumulation in the stamen envelope, occurring during meiosis and pollen mitosis I, we identified a novel, third wave of starch amylogenesis/amylolysis during the last stages of stamen development. To gain insights into the underlying molecular mechanisms, we analyzed publicly available microarray data, which revealed a developmentally coordinated expression of carbohydrate transport and metabolism genes during these waves of transient starch accumulation. Based on this analysis, we characterized starch dynamics in mutants affecting hexose phosphate metabolism and translocation, and identified the Glc-6-phosphate/phosphate antiporter GPT1 as the putative translocator of Glc-6-phosphate for starch biosynthesis in reproductive tissues. Based on these results, we propose a model of starch synthesis within the pollen grain and discuss the nutrient transport route feeding the embryo within the developing seed.

Rethinking Guard Cell Metabolism.

Stomata control gaseous fluxes between the internal leaf air spaces and the external atmosphere and, therefore, play a pivotal role in regulating CO2 uptake for photosynthesis as well as water loss through transpiration. Guard cells, which flank the stomata, undergo adjustments in volume, resulting in changes in pore aperture. Stomatal opening is mediated by the complex regulation of ion transport and solute biosynthesis. Ion transport is exceptionally well understood, whereas our knowledge of guard cell metabolism remains limited, despite several decades of research. In this review, we evaluate the current literature on metabolism in guard cells, particularly the roles of starch, sucrose, and malate. We explore the possible origins of sucrose, including guard cell photosynthesis, and discuss new evidence that points to multiple processes and plasticity in guard cell metabolism that enable these cells to function effectively to maintain optimal stomatal aperture. We also discuss the new tools, techniques, and approaches available for further exploring and potentially manipulating guard cell metabolism to improve plant water use and productivity.

Regulation of Leaf Starch Degradation by Abscisic Acid Is Important for Osmotic Stress Tolerance in Plants.

Starch serves functions that range over a timescale of minutes to years, according to the cell type from which it is derived. In guard cells, starch is rapidly mobilized by the synergistic action of ß-AMYLASE1 (BAM1) and α-AMYLASE3 (AMY3) to promote stomatal opening. In the leaves, starch typically accumulates gradually during the day and is degraded at night by BAM3 to support heterotrophic metabolism. During osmotic stress, starch is degraded in the light by stress-activated BAM1 to release sugar and sugar-derived osmolytes. Here, we report that AMY3 is also involved in stress-induced starch degradation. Recently isolated Arabidopsis thaliana amy3 bam1 double mutants are hypersensitive to osmotic stress, showing impaired root growth. amy3 bam1 plants close their stomata under osmotic stress at similar rates as the wild type but fail to mobilize starch in the leaves. (14)C labeling showed that amy3 bam1 plants have reduced carbon export to the root, affecting osmolyte accumulation and root growth during stress. Using genetic approaches, we further demonstrate that abscisic acid controls the activity of BAM1 and AMY3 in leaves under osmotic stress through the AREB/ABF-SnRK2 kinase-signaling pathway. We propose that differential regulation and isoform subfunctionalization define starch-adaptive plasticity, ensuring an optimal carbon supply for continued growth under an ever-changing environment.

ß-amylase 1 (BAM1) degrades transitory starch to sustain proline biosynthesis during drought stress.

During photosynthesis of higher plants, absorbed light energy is converted into chemical energy that, in part, is accumulated in the form of transitory starch within chloroplasts. In the following night, transitory starch is mobilized to sustain the heterotrophic metabolism of the plant. ß-amylases are glucan hydrolases that cleave α-1,4-glycosidic bonds of starch and release maltose units from the non-reducing end of the polysaccharide chain. In Arabidopsis, nocturnal degradation of transitory starch involves mainly ß-amylase-3 (BAM3). A second ß-amylase isoform, ß-amylase-1 (BAM1), is involved in diurnal starch degradation in guard cells, a process that sustains stomata opening. However, BAM1 also contributes to diurnal starch turnover in mesophyll cells under osmotic stress. With the aim of dissecting the role of ß-amylases in osmotic stress responses in Arabidopsis, mutant plants lacking either BAM1 or BAM3 were subject to a mild (150mM mannitol) and prolonged (up to one week) osmotic stress. We show here that leaves of osmotically-stressed bam1 plants accumulated more starch and fewer soluble sugars than both wild-type and bam3 plants during the day. Moreover, bam1 mutants were impaired in proline accumulation and suffered from stronger lipid peroxidation, compared with both wild-type and bam3 plants. Taken together, these data strongly suggest that carbon skeletons deriving from BAM1 diurnal degradation of transitory starch support the biosynthesis of proline required to face the osmotic stress. We propose the transitory-starch/proline interplay as an interesting trait to be tackled by breeding technologies aimingto improve drought tolerance in relevant crops.