Chilling induces sugar and ABA accumulation that antagonistically signals for symplastic connection of dormant potato buds.

Endodormancy (ED) is a crucial stage in the life cycle of many perennial plants. ED release requires accumulating a certain amount of cold exposure, measured as chilling units. However, the mechanism governing the effect of chilling on ED duration is poorly understood. We used the potato tuber model to investigate the response to chilling as associated with ED release. We measured the accumulation of specific sugars during and after chilling, defined as sugar units. We discovered that ED duration correlated better with sugar units accumulation than chilling units. A logistic function was developed based on sugar units measurements to predict ED duration. Knockout or overexpression of the vacuolar invertase gene (StVInv) unexpectedly modified sugar units levels and extended or shortened ED, respectively. Silencing the energy sensor SNF1-related protein kinase 1, induced higher sugar units accumulation and shorter ED. Sugar units accumulation induced by chilling or transgenic lines reduced plasmodesmal (PD) closure in the dormant bud meristem. Chilling or knockout of abscisic acid (ABA) 8'-hydroxylase induced ABA accumulation, in parallel to sweetening, and antagonistically promoted PD closure. Our results suggest that chilling induce sugar units and ABA accumulation, resulting in antagonistic signals for symplastic connection of the dormant bud.

Guard-cell-targeted overexpression of Arabidopsis Hexokinase 1 can improve water use efficiency in field-grown tobacco plants.

Water deficit currently acts as one of the largest limiting factors for agricultural productivity worldwide. Additionally, limitation by water scarcity is projected to continue in the future with the further onset of effects of global climate change. As a result, it is critical to develop or breed for crops that have increased water use efficiency and that are more capable of coping with water scarce conditions. However, increased intrinsic water use efficiency (iWUE) typically brings a trade-off with CO2 assimilation as all gas exchange is mediated by stomata, through which CO2 enters the leaf while water vapor exits. Previously, promising results were shown using guard-cell-targeted overexpression of hexokinase to increase iWUE without incurring a penalty in photosynthetic rates or biomass production. Here, two homozygous transgenic tobacco (Nicotiana tabacum) lines expressing Arabidopsis Hexokinase 1 (AtHXK1) constitutively (35SHXK2 and 35SHXK5) and a line that had guard-cell-targeted overexpression of AtHXK1 (GCHXK2) were evaluated relative to wild type for traits related to photosynthesis and yield. In this study, iWUE was significantly higher in GCHXK2 compared with wild type without negatively impacting CO2 assimilation, although results were dependent upon leaf age and proximity of precipitation event to gas exchange measurement.

Sugar and hexokinase suppress expression of PIP aquaporins and reduce leaf hydraulics that preserves leaf water potential.

Sugars affect central aspects of plant physiology, including photosynthesis, stomatal behavior and the loss of water through the stomata. Yet, the potential effects of sugars on plant aquaporins (AQPs) and water conductance have not been examined. We used database and transcriptional analyses, as well as cellular and whole-plant functional techniques to examine the link between sugar-related genes and AQPs. Database analyses revealed a high level of correlation between the expression of AQPs and that of sugar-related genes, including the Arabidopsis hexokinases 1 (AtHXK1). Increased expression of AtHXK1, as well as the addition of its primary substrate, glucose (Glc), repressed the expression of 10 AQPs from the plasma membrane-intrinsic proteins (PIP) subfamily (PIP-AQPs) and induced the expression of two stress-related PIP-AQPs. The osmotic water permeability of mesophyll protoplasts of AtHXK1-expressing plants and the leaf hydraulic conductance of those plants were significantly reduced, in line with the decreased expression of PIP-AQPs. Conversely, hxk1 mutants demonstrated a higher level of hydraulic conductance, with increased water potential in their leaves. In addition, the presence of Glc reduced leaf water potential, as compared with an osmotic control, indicating that Glc reduces the movement of water from the xylem into the mesophyll. The production of sugars entails a significant loss of water and these results suggest that sugars and AtHXK1 affect the expression of AQP genes and reduce leaf water conductance, to coordinate sugar levels with the loss of water through transpiration.

The Solanum tuberosum KST1 partial promoter as a tool for guard cell expression in multiple plant species.

To date, guard cell promoters have been examined in only a few species, primarily annual dicots. A partial segment of the potato (Solanum tuberosum) KST1 promoter (KST1 partial promoter, KST1ppro) has previously been shown to confer guard cell expression in potato, tomato (Solanum lycopersicum), citrus [Troyer citrange (C. sinensis×Poncirus trifoliata)], and Arabidopsis (Arabidopsis thaliana). Here, we describe an extensive analysis of the expression pattern of KST1ppro in eight (previously reported, as well as new) species from five different angiosperm families, including the Solanaceae and the Cucurbitaceae, Arabidopsis, the monocot barley (Hordeum vulgare), and two perennial species: grapevine (Vitis vinifera) and citrus. Using confocal imaging and three-dimensional movies, we demonstrate that KST1ppro drives guard cell expression in all of these species, making it the first dicot-originated guard cell promoter shown to be active in a monocot and the first promoter reported to confer guard cell expression in barley and cucumber (Cucumis sativus). The results presented here indicate that KST1ppro can be used to drive constitutive guard cell expression in monocots and dicots and in both annual and perennial plants. In addition, we show that the KST1ppro is active in guard cells shortly after the symmetric division of the guard mother cell and generates stable expression in mature guard cells. This allows us to follow the spatial and temporal distribution of stomata in cotyledons and true leaves.

The tomato plastidic fructokinase SlFRK3 plays a role in xylem development.

Plants have two kinds of fructokinases (FRKs) that catalyze the key step of fructose phosphorylation, cytosolic and plastidic. The major cytosolic tomato FRK, SlFRK2, is essential for the development of xylem vessels. In order to study the role of SlFRK3, which encodes the only plastidic FRK, we generated transgenic tomato (Solanum lycopersicon) plants with RNAi suppression of SlFRK3 as well as plants expressing beta-glucoronidase (GUS) under the SlFRK3 promoter. GUS staining indicated SlFRK3 expression in vascular tissues of the leaves and stems, including cambium, differentiating xylem, young xylem fibers and phloem companion cells. Suppression of SlFRK3 reduced the stem xylem area, stem and root water conductance, and whole-plant transpiration, with minor effects on plant development. However, suppression of SlFRK3 accompanied by partial suppression of SlFRK2 induced significant growth-inhibition effects, including the wilting of mature leaves. Grafting experiments revealed that these growth effects are imposed primarily by the leaves, whose petioles had unlignified, thin-walled xylem fibers with collapsed parenchyma cells around the vessels. A cross between the SlFRK2-antisense and SlFRK3-RNAi lines exhibited similar wilting and anatomical effects, confirming that these effects are the result of the combined suppression of SlFRK3 and SlFRK2. These results demonstrate a role of the plastidic SlFRK3 in xylem development and hydraulic conductance.

The role of plasma membrane aquaporins in regulating the bundle sheath-mesophyll continuum and leaf hydraulics.

Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydraulics, in general, and extravascular, radial hydraulic conductance in leaves (K(leaf)), in particular, is still fairly limited. We hypothesized that the AQPs of the vascular bundle sheath (BS) cells regulate K(leaf). To examine this hypothesis, AQP genes were silenced using artificial microRNAs that were expressed constitutively or specifically targeted to the BS. MicroRNA sequences were designed to target all five AQP genes from the PLASMA MEMBRANE-INTRINSIC PROTEIN1 (PIP1) subfamily. Our results show that the constitutively silenced PIP1 (35S promoter) plants had decreased PIP1 transcript and protein levels and decreased mesophyll and BS osmotic water permeability (P(f)), mesophyll conductance of CO2, photosynthesis, K(leaf), transpiration, and shoot biomass. Plants in which the PIP1 subfamily was silenced only in the BS (SCARECROW:microRNA plants) exhibited decreased mesophyll and BS Pf and decreased K(leaf) but no decreases in the rest of the parameters listed above, with the net result of increased shoot biomass. We excluded the possibility of SCARECROW promoter activity in the mesophyll. Hence, the fact that SCARECROW:microRNA mesophyll exhibited reduced P(f), but not reduced mesophyll conductance of CO2, suggests that the BS-mesophyll hydraulic continuum acts as a feed-forward control signal. The role of AQPs in the hierarchy of the hydraulic signal pathway controlling leaf water status under normal and limited-water conditions is discussed.

Hexokinase mediates stomatal closure.

Stomata, composed of two guard cells, are the gates whose controlled movement allows the plant to balance the demand for CO2 for photosynthesis with the loss of water through transpiration. Increased guard-cell osmolarity leads to the opening of the stomata and decreased osmolarity causes the stomata to close. The role of sugars in the regulation of stomata is not yet clear. In this study, we examined the role of hexokinase (HXK), a sugar-phosphorylating enzyme involved in sugar-sensing, in guard cells and its effect on stomatal aperture. We show here that increased expression of HXK in guard cells accelerates stomatal closure. We further show that this closure is induced by sugar and is mediated by abscisic acid. These findings support the existence of a feedback-inhibition mechanism that is mediated by a product of photosynthesis, namely sucrose. When the rate of sucrose production exceeds the rate at which sucrose is loaded into the phloem, the surplus sucrose is carried toward the stomata by the transpiration stream and stimulates stomatal closure via HXK, thereby preventing the loss of precious water.

Mesophyll photosynthesis and guard cell metabolism impacts on stomatal behaviour.

Stomata control gaseous fluxes between the internal leaf air spaces and the external atmosphere. Guard cells determine stomatal aperture and must operate to ensure an appropriate balance between CO2 uptake for photosynthesis (A) and water loss, and ultimately plant water use efficiency (WUE). A strong correlation between A and stomatal conductance (gs ) is well documented and often observed, but the underlying mechanisms, possible signals and metabolites that promote this relationship are currently unknown. In this review we evaluate the current literature on mesophyll-driven signals that may coordinate stomatal behaviour with mesophyll carbon assimilation. We explore a possible role of various metabolites including sucrose and malate (from several potential sources; including guard cell photosynthesis) and new evidence that improvements in WUE have been made by manipulating sucrose metabolism within the guard cells. Finally we discuss the new tools and techniques available for potentially manipulating cell-specific metabolism, including guard and mesophyll cells, in order to elucidate mesophyll-derived signals that coordinate mesophyll CO2 demands with stomatal behaviour, in order to provide a mechanistic understanding of these processes as this may identify potential targets for manipulations in order to improve plant WUE and crop yield.

Substantial roles of hexokinase and fructokinase in the effects of sugars on plant physiology and development.

The basic requirements for plant growth are light, CO2, water, and minerals. However, the absorption and utilization of each of these requires investment on the part of the plant. The primary products of plants are sugars, and the hexose sugars glucose and fructose are the raw material for most of the metabolic pathways and organic matter in plants. To be metabolized, hexose sugars must first be phosphorylated. Only two families of enzymes capable of catalysing the essential irreversible phosphorylation of glucose and fructose have been identified in plants, hexokinases (HXKs) and fructokinases (FRKs). These hexose-phosphorylating enzymes appear to coordinate sugar production with the abilities to absorb light, CO2, water, and minerals. This review describes the long- and short-term effects mediated by HXK and FRK in various tissues, as well as the role of these enzymes in the coordination of sugar production with the absorption of light, CO2, water, and minerals.

Guard cell activity of PIF4 and HY5 control transpiration.

Whole-plant transpiration, controlled by plant hydraulics and stomatal movement, is regulated by endogenous and environmental signals, with the light playing a dominant role. Stomatal pore size continuously adjusts to changes in light intensity and quality to ensure optimal CO2 intake for photosynthesis on the one hand, together with minimal water loss on the other. The link between light and transpiration is well established, but the genetic knowledge of how guard cells perceive those signals to affect stomatal conductance is still somewhat limited. In the current study, we evaluated the role of two central light-responsive transcription factors; a bZIP-family transcription factor ELONGATED HYPOCOTYL5 (HY5) and the basic helix-loop-helix (BHLH) transcription factor PHYTOCHROME INTERACTING FACTOR4 (PIF4), in the regulation of steady-state transpiration. We show that overexpression of PIF4 exclusively in guard cells (GCPIF4) decreases transpiration, and can restrain the high transpiration of the pif4 mutant. Expression of HY5 specifically in guard cells (GCHY5) had the opposite effect of enhancing transpiration rates of WT- Arabidopsis and tobacco plants and of the hy5 mutant in Arabidopsis. In addition, we show that GCHY5 can reverse the low transpiration caused by guard cell overexpression of the sugar sensor HEXOKINASE1 (HXK1, GCHXK), an established low transpiring genotype. Finally, we suggest that the GCHY5 reversion of low transpiration by GCHXK requires the auto-activation of the endogenous HY5 in other tissues. These findings support the existence of an ongoing diurnal regulation of transpiration by the light-responsive transcription factors HY5 and PIF4 in the stomata, which ultimately determine the whole-plant water use efficiency.

A plant small polypeptide is a novel component of DNA-binding protein phosphatase 1-mediated resistance to plum pox virus in Arabidopsis.

DNA-binding protein phosphatases (DBPs) have been identified as a novel class of plant-specific regulatory factors playing a role in plant-virus interactions. NtDBP1 from tobacco (Nicotiana tabacum) was shown to participate in transcriptional regulation of gene expression in response to virus infection in compatible interactions, and AtDBP1, its closest relative in the model plant Arabidopsis (Arabidopsis thaliana), has recently been found to mediate susceptibility to potyvirus, one of the most speciose taxa of plant viruses. Here, we report on the identification of a novel family of highly conserved small polypeptides that interact with DBP1 proteins both in tobacco and Arabidopsis, which we have designated DBP-interacting protein 2 (DIP2). The interaction of AtDIP2 with AtDBP1 was demonstrated in vivo by bimolecular fluorescence complementation, and AtDIP2 was shown to functionally interfere with AtDBP1 in yeast. Furthermore, reducing AtDIP2 gene expression leads to increased susceptibility to the potyvirus Plum pox virus and to a lesser extent also to Turnip mosaic virus, whereas overexpression results in enhanced resistance. Therefore, we describe a novel family of conserved small polypeptides in plants and identify AtDIP2 as a novel host factor contributing to resistance to potyvirus in Arabidopsis.

Sucrose Synthase and Fructokinase Are Required for Proper Meristematic and Vascular Development.

Sucrose synthase (SuSy) and fructokinase (FRK) work together to control carbohydrate flux in sink tissues. SuSy cleaves sucrose into fructose and UDP-glucose; whereas FRK phosphorylates fructose. Previous results have shown that suppression of the SUS1,3&4 genes by SUS-RNAi alters auxin transport in the shoot apical meristems of tomato plants and affects cotyledons and leaf structure; whereas antisense suppression of FRK2 affects vascular development. To explore the joint developmental roles of SuSy and FRK, we crossed SUS-RNAi plants with FRK2-antisense plants to create double-mutant plants. The double-mutant plants exhibited novel phenotypes that were absent from the parent lines. About a third of the plants showed arrested shoot apical meristem around the transition to flowering and developed ectopic meristems. Use of the auxin reporter DR5::VENUS revealed a significantly reduced auxin response in the shoot apical meristems of the double-mutant, indicating that auxin levels were low. Altered inflorescence phyllotaxis and significant disorientation of vascular tissues were also observed. In addition, the fruits and the seeds of the double-mutant plants were very small and the seeds had very low germination rates. These results show that SUS1,3&4 and FRK2 enzymes are jointly essential for proper meristematic and vascular development, and for fruit and seed development.

Metabolism of soluble sugars in developing melon fruit: a global transcriptional view of the metabolic transition to sucrose accumulation.

The sweet melon fruit is characterized by a metabolic transition during its development that leads to extensive accumulation of the disaccharide sucrose in the mature fruit. While the biochemistry of the sugar metabolism pathway of the cucurbits has been well studied, a comprehensive analysis of the pathway at the transcriptional level allows for a global genomic view of sugar metabolism during fruit sink development. We identified 42 genes encoding the enzymatic reactions of the sugar metabolism pathway in melon. The expression pattern of the 42 genes during fruit development of the sweet melon cv Dulce was determined from a deep sequencing analysis performed by 454 pyrosequencing technology, comprising over 350,000 transcripts from four stages of developing melon fruit flesh, allowing for digital expression of the complete metabolic pathway. The results shed light on the transcriptional control of sugar metabolism in the developing sweet melon fruit, particularly the metabolic transition to sucrose accumulation, and point to a concerted metabolic transition that occurs during fruit development.

Comparison of a novel tomato sucrose synthase, SlSUS4, with previously described SlSUS isoforms reveals distinct sequence features and differential expression patterns in association with stem maturation.

Sucrose synthase (SUS) plays a role in many contexts of sugar metabolism, including low-oxygen and low-ATP respiration and the synthesis of cellulose. In tomato (Solanum lycopersicum), as in many plants, SUS is encoded by genes at several independent loci. Here, we report the isolation of a novel tomato SUS (SlSUS) isoform, SlSUS4, that is homologous to potato SUS isoform 1 (StSUS1) and also shows greater homology to SUS isoforms of other plants than to the other tomato SUS isoforms. All three tomato isoforms are very similar in genomic structure and sequence, yet each is located on a separate chromosome. Real-time expression analysis of the three distinct isoforms revealed widely varying patterns of expression, in terms of both tissue specificity and overall magnitude of expression. Analysis of SlSUS expression along the tomato stem revealed opposing expression gradients for two of the SlSUS isoforms, in apparent correlation with vascular tissue maturation. Western-blot analysis of SlSUS protein showed an increasing SlSUS concentration gradient along the developmental axis of the tomato stem, with the protein concentrated mainly in the vascular tissue of the stem. These gene expression and protein accumulation patterns indicate that each isoform may play a discrete role in the development of tomato plants, most notably in the development of vascular tissue in the stem.

Complex chromosomal rearrangement in a girl with psychomotor-retardation and a de novo inversion: inv(2)(p15;q24.2).

Cytogenetic analysis of DNA from a girl with severe psychomotor retardation revealed a de novo pericentric inversion of chromosome 2: 46,XX,inv(2)(p15q24.2). In order to elucidate the possible role of the inversion in the girl's abnormal phenotype, we analyzed the inversion breakpoints. FISH analysis revealed BAC clones spanning the breakpoints at 2p and 2q of the inversion. Southern blot hybridization with DNA probes from the BAC regions was used to refine the localization of the breakpoints, followed by inverse-PCR which enabled us to sequence the inversion breakpoints. We found a complex chromosomal rearrangement, including five breakpoints, four at 2q and one at 2p joined with minor insertions/deletions of a few bases. The breakpoint at 2p was within the NRXN1 gene that has previously been associated with autism, intellectual disabilities, and psychiatric disorders. In 2q, the breakpoints disrupted two genes, TANC1 and RBMS1; the phenotypic effect of these genes is not currently known.

Guard cells control hypocotyl elongation through HXK1, HY5, and PIF4.

The hypocotyls of germinating seedlings elongate in a search for light to enable autotrophic sugar production. Upon exposure to light, photoreceptors that are activated by blue and red light halt elongation by preventing the degradation of the hypocotyl-elongation inhibitor HY5 and by inhibiting the activity of the elongation-promoting transcription factors PIFs. The question of how sugar affects hypocotyl elongation and which cell types stimulate and stop that elongation remains unresolved. We found that overexpression of a sugar sensor, Arabidopsis hexokinase 1 (HXK1), in guard cells promotes hypocotyl elongation under white and blue light through PIF4. Furthermore, expression of PIF4 in guard cells is sufficient to promote hypocotyl elongation in the light, while expression of HY5 in guard cells is sufficient to inhibit the elongation of the hy5 mutant and the elongation stimulated by HXK1. HY5 exits the guard cells and inhibits hypocotyl elongation, but is degraded in the dark. We also show that the inhibition of hypocotyl elongation by guard cells' HY5 involves auto-activation of HY5 expression in other tissues. It appears that guard cells are capable of coordinating hypocotyl elongation and that sugar and HXK1 have the opposite effect of light on hypocotyl elongation, converging at PIF4.

Future-Proofing Potato for Drought and Heat Tolerance by Overexpression of Hexokinase and SP6A.

Crop yield is largely affected by global climate change. Especially periods of heat and drought limit crop productivity worldwide. According to current models of future climate scenarios, heatwaves and periods of drought are likely to increase. Potato, as an important food crop of temperate latitudes, is very sensitive to heat and drought which impact tuber yield and quality. To improve abiotic stress resilience of potato plants, we aimed at co-expressing hexokinase 1 from Arabidopsis thaliana (AtHXK1) in guard cells and SELF-PRUNING 6A (SP6A) using the leaf/stem-specific StLS1 promoter in order to increase water use efficiency as well as tuberization under drought and heat stress. Guard cell-specific expression of AtHXK1 decreased stomatal conductance and improved water use efficiency of transgenic potato plants as has been shown for other crop plants. Additionally, co-expression with the FT-homolog SP6A stimulated tuberization and improved assimilate allocation to developing tubers under control as well as under single and combined drought and heat stress conditions. Thus, co-expression of both proteins provides a novel strategy to improve abiotic stress tolerance of potato plants.

Expression of Hexokinase in Stomata of Citrus Fruit Reduces Fruit Transpiration and Affects Seed Development.

The temporal formation and spatial distribution of stomata on the surface of citrus floral organs and, specifically, on the ovule from which the fruit develops, were analyzed using citrus plants that express green fluorescent protein (GFP) under the guard cell-specific KST1 promoter. Stomata are found on the style, sepal, and anther of the closed flower and on ovules from the stage of anthesis. It has previously been shown that hexokinase (HXK) mediates sugar-sensing in leaf guard cells and stimulates stomatal closure. The activity and response of citrus fruit stomata to sugar-sensing by HXK was examined using plants that express HXK under the KST1 promoter. Those plants are referred to as GCHXK plants. The transpiration of young green GCHXK citrus fruits was significantly reduced, indicating that their stomata respond to sugar similar to leaf stomata. Toward fruit maturation, fruit stomata are plugged and stop functioning, which explains why WT and GCHXK mature yellow fruits exhibited similar water loss. Seeds of the GCHXK plants were smaller and germinated more slowly than the WT seeds. We suggest that the stomata of young green citrus fruits, but not mature yellow fruits, respond to sugar levels via HXK and that fruit stomata are important for proper seed development.

LeFRK2 is required for phloem and xylem differentiation and the transport of both sugar and water.

It has been suggested that LeFRK2, the major fructose-phosphorylating enzyme in tomato plants, may be required for stem xylem development. Yet, we do not know if this enzyme affects the development of individual vessels, whether it affects water conductance, or whether it affects phloem development and sugar transport. Here, we show that suppression of LeFRK2 results in a significant reduction in the size of vascular cells and slows fiber maturation. The vessels in stems of LeFRK2-antisense plants are narrower than in WT plants and have thinner secondary cell walls. Although the cambium produces rounded secondary vessels, these vessels become deformed during the early stages of xylem maturation. Water conductance is then reduced in stems, roots, and leaves, suggesting that LeFRK2 influences xylem development throughout the entire vascular system. Interestingly, the build-up of positive xylem pressure under static (no-flow) conditions was also decreased. Suppression of LeFRK2 reduced the length and width of the sieve elements, as well as callose deposition. To examine the effect of LeFRK2 suppression on phloem transport, we created triple-grafted plants in which a portion of the wild-type stem was replaced with an antisense interstcok, and compared the contents of the transported sugar, sucrose, in the different portions of these stems. Sucrose contents above and within the LeFRK2-antisense interstock were significantly higher than those below the graft. These results show that the antisense interstock restricted the downward movement of sucrose, suggesting that LeFRK2 is required for both phloem and xylem development.