Guard cells represent a unique single cell-type system for the study of cellular responses to abi... more Guard cells represent a unique single cell-type system for the study of cellular responses to abiotic and biotic perturbations that affect stomatal movement. Decades of effort through both classical physiological and functional genomics approaches have generated an enormous amount of information on the roles of individual metabolites in stomatal guard cell function and physiology. Recent application of metabolomics methods has produced a substantial amount of new information on metabolome control of stomatal movement. In conjunction with other "omics" approaches, the knowledge-base is growing to reach a systems-level description of this single cell-type. Here we summarize current knowledge of the guard cell metabolome and highlight critical metabolites that bear significant impact on future engineering and breeding efforts to generate plants/crops that are resistant to environmental challenges and produce high yield and quality products for food and energy security.
Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydr... more 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.
Stomata, composed of two guard cells, are the gates whose controlled movement allows the plant to... more 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.
Increased expression of the aquaporin NtAQP1, which is known to function as a plasmalemma channel... more Increased expression of the aquaporin NtAQP1, which is known to function as a plasmalemma channel for CO 2 and water, increases the rate of both photosynthesis and transpiration. In contrast, increased expression of Arabidopsis hexokinase1 (AtHXK1), a dual-function enzyme that mediates sugar sensing, decreases the expression of photosynthetic genes and the rate of transpiration and inhibits growth. Here, we show that AtHXK1 also decreases root and stem hydraulic conductivity and leaf mesophyll CO 2 conductance (g m ). Due to their opposite effects on plant development and physiology, we examined the relationship between NtAQP1 and AtHXK1 at the whole-plant level using transgenic tomato plants expressing both genes simultaneously. NtAQP1 significantly improved growth and increased the transpiration rates of AtHXK1expressing plants. Reciprocal grafting experiments indicated that this complementation occurs when both genes are expressed simultaneously in the shoot. Yet, NtAQP1 had only a marginal effect on the hydraulic conductivity of the doubletransgenic plants, suggesting that the complementary effect of NtAQP1 is unrelated to shoot water transport. Rather, NtAQP1 significantly increased leaf mesophyll CO 2 conductance and enhanced the rate of photosynthesis, suggesting that NtAQP1 facilitated the growth of the double-transgenic plants by enhancing mesophyll conductance of CO 2 .
Two different partial sequences encoding putative hexokinase (HXK, ATP: hexose-6-phosphotransfera... more Two different partial sequences encoding putative hexokinase (HXK, ATP: hexose-6-phosphotransferase, EC 2.7.1.1) were isolated from tomato (Lycopersicon esculentum) by RT-PCR using degenerate primers. Southern blot analysis suggested the existence of two divergent HXK genes. A complete cDNA of one HXK was isolated by screening a cDNA library prepared from young cherry tomato fruit. The 1770 bp cDNA of LeHXK2 contained
The LeFRK2 gene product has been proposed to play a role in transient starch accumulation in deve... more The LeFRK2 gene product has been proposed to play a role in transient starch accumulation in developing tomato (Lycopersicon esculentum) fruits. To analyze the role of LeFRK2 in starch biosynthesis, we produced transgenic tomato plants with sense and antisense expression of StFRK, the potato homologue to LeFRK2. Fruits of homozygous plants expressing sense or antisense StFRK exhibited suppression of LeFRK2, concomitantly with specific elimination of the FKI isozyme, indicating that FKI is the gene product of LeFRK2. The activity of fructokinase was reduced in antisense and sense homozygous fruits and increased in fruits of sense hemizygous plants. The modified activities of fructokinase led to small but significant changes both in the steady state levels of sugars and in the level of phosphorylated sugars in fruits. However, fruits lacking FKI had increased rather than decreased starch content. We therefore concluded that LeFRK2 is not required for transient starch accumulation in tomato fruits. Rather, LeFRK2 might have a role in the regulation of sucrose import into tomato fruits.
A full-length cDNA encoding a novel fourth fructokinase, LeFRK3, was cloned from green tomato (Ly... more A full-length cDNA encoding a novel fourth fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate
The segregation of the nucleolus during mitosis was examined in Saccharomyces cerevisiae and Schi... more The segregation of the nucleolus during mitosis was examined in Saccharomyces cerevisiae and Schizosaccharomyces pombe by indirect immunofluorescence using antibodies directed to highly conserved anti-nucleolus antigens. In mitotic S. pombe cells, the nucleolus appears to trail the bulk of the DNA. In wild-type cells of S. cerevisiae, the nucleolus segregates alongside the bulk of the genomic DNA. Based on its distance from the centromere, we would expect the rDNA in both organisms to segregate behind the majority of the genomic DNA, if telomeric regions trail centromeric regions as in other eukaryotes. We therefore suggest that in S. cerevisiae the nucleolus is attached to other parts of the nucleus which enable it to segregate along with the bulk of the DNA. The segregation of the nucleolus in topoisomerase mutants and nuclear division mutants of S. cerevisiae was also investigated. In cdc14 mutants which arrest at late anaphase, the vast majority of the DNA is separated, but the ...
Carbohydrates are essential for male gametophyte development. However, our understanding of the m... more Carbohydrates are essential for male gametophyte development. However, our understanding of the mechanism by which the sugar supply is controlled in the stamen is still in its infancy. We previously reported on the stamen-specific expression of the tomato (Solanum lycopersicum) sugar metabolic gene, fructokinase 4 (SlFRK4). Here, we present the cloning and the characterization of the SlFRK4 promoter and show its differential activation during anther development. We also show that the tissue-specific expression of SlFRK4 promoter is maintained in Arabidopsis thaliana. By histochemical analyses of the GUS reporter gene and DTA toxin driven by the SlFRK4 promoter, we show that the SlFRK4 promoter is gradually activated in pollen grains throughout the later stages of anther development and upon pollen germination. In addition, we analyzed the expression profile of SlFRK4 and other sugar metabolic genes and found that SlFRK4 and the invertase LIN7 are co-expressed in mature and germinated pollen. These findings point to the existence of a specialized mechanism in which carbohydrates are provided to the male gametophyte during the later stages of its development and suggest a valuable tool for manipulating the development of male gametophytes in crop species.
Increased expression of the aquaporin NtAQP1, which is known to function as a plasmalemma channel... more Increased expression of the aquaporin NtAQP1, which is known to function as a plasmalemma channel for CO 2 and water, increases the rate of both photosynthesis and transpiration. In contrast, increased expression of Arabidopsis hexokinase1 (AtHXK1), a dual-function enzyme that mediates sugar sensing, decreases the expression of photosynthetic genes and the rate of transpiration and inhibits growth. Here, we show that AtHXK1 also decreases root and stem hydraulic conductivity and leaf mesophyll CO 2 conductance (g m ). Due to their opposite effects on plant development and physiology, we examined the relationship between NtAQP1 and AtHXK1 at the whole-plant level using transgenic tomato plants expressing both genes simultaneously. NtAQP1 significantly improved growth and increased the transpiration rates of AtHXK1expressing plants. Reciprocal grafting experiments indicated that this complementation occurs when both genes are expressed simultaneously in the shoot. Yet, NtAQP1 had only a marginal effect on the hydraulic conductivity of the doubletransgenic plants, suggesting that the complementary effect of NtAQP1 is unrelated to shoot water transport. Rather, NtAQP1 significantly increased leaf mesophyll CO 2 conductance and enhanced the rate of photosynthesis, suggesting that NtAQP1 facilitated the growth of the double-transgenic plants by enhancing mesophyll conductance of CO 2 .
Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydr... more 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.
Hexose sugars, such as glucose and fructose produced in plants, are ubiquitous in most organisms ... more Hexose sugars, such as glucose and fructose produced in plants, are ubiquitous in most organisms and are the origin of most of the organic matter found in nature. To be utilized, hexose sugars must first be phosphorylated. The central role of hexose-phosphorylating enzymes has attracted the attention of many researchers, leading to novel discoveries. Only two families of enzymes capable of phosphorylating glucose and fructose have been identified in plants; hexokinases (HXKs), and fructokinases (FRKs). Intensive investigations of these two families in numerous plant species have yielded a wealth of knowledge regarding the genes number, enzymatic characterization, intracellular localization, and developmental and physiological roles of several HXKs and FRKs. The emerging picture indicates that HXK and FRK enzymes found at specific intracellular locations play distinct roles in plant metabolism and development. Individual HXKs were shown for the first time to be dual-function enzymes -sensing sugar levels independent of their catalytic activity and controlling gene expression and major developmental pathways, as well as hormonal interactions. FRK, on the other hand, seems to play a central metabolic role in vascular tissues, controlling the amounts of sugars allocated for vascular development. While a clearer picture of the roles of these two types of enzymes is emerging, many questions remain unsolved, such as the specific tissues and types of cells in which these enzymes function, the roles of individual HXK and FRK genes, and how these enzymes interact with hormones in the regulation of developmental processes. It is anticipated that ongoing efforts will broaden our knowledge of these important plant enzymes and their potential uses in the modification of plant traits.
Hexokinases (HXKs), catalysts of the first essential step in glucose metabolism, have emerged as ... more Hexokinases (HXKs), catalysts of the first essential step in glucose metabolism, have emerged as important enzymes that mediate sugar sensing in many organisms, including plants. The presence of several types of plant HXK isozymes, located in different intracellular locations, has been suggested. However, recent studies have indicated that most plants have only two types of HXKs, a plastidic stromal isozyme and membrane-associated isozymes located mainly adjacent to the mitochondria, but also in the nucleus. The membrane-associated isozymes are involved in sugar sensing and regulate gene expression. The central role of HXKs in plant development and the increasing interest in their role necessitate the correction of inaccuracies that have spread concerning the substrate specificity and intracellular localization of HXK isozymes, as these inaccuracies are affecting the hypothesized roles presented for these isozymes and shaping future research in this active field.
Sarcotoxin IA is a cecropin-type antibacterial protein produced by the flesh fly,Sarcophaga pereg... more Sarcotoxin IA is a cecropin-type antibacterial protein produced by the flesh fly,Sarcophaga peregrina.Similar to other bactericidal small proteins produced by insects, sarcotoxin IA is released into the hemolymph of larvae and nymphs upon mechanical injury or bacterial infection. The gene (sarco) that encodes this toxin was introduced intoSaccharomyces cerevisiaeyeast cells and was expressed under a constitutive yeast promoter. The transformed
By using immunolocalization and differential extraction methods we show that only apoplastic inve... more By using immunolocalization and differential extraction methods we show that only apoplastic invertase, but not vacuolar invertase, was present in the mature, sucrose-accumulating L. hirsutum pericarp. In contrast, in the hexoseaccumulating L. esculentum fruit, both the apoplastic and vacuolar invertase activities and protein content increase in the mature fruit. Quantitative expression studies of the soluble invertase gene (TIV1) and the apoplastic invertase genes (LINs) showed that only TIV1 gene expression could account for the species and developmental differences of both soluble and insoluble enzyme activity of the pericarp. The expression of the LIN genes encoding for apoplastic tomato invertases was unrelated to the differences in bound enzyme activity and
Two divergent genes encoding fructokinase, Frk1 and Frk2, have been previously shown to be expres... more Two divergent genes encoding fructokinase, Frk1 and Frk2, have been previously shown to be expressed in tomato (Lycopersicon esculentum L.) and have now been further characterized with re- gard to their spatial expression and the enzymic properties of the encoded proteins. Frk1 and Frk2 mRNA levels were coordinately induced by exogenous sugar, indicating that both belong to the growing class of sugar-regulated genes. However, in situ hybridiza- tion indicated that Frk1 and Frk2 were expressed in a spatially distinct manner, with Frk2 mRNA primarily localized in cells of the fruit pericarp, which store starch, and Frk1 mRNA distributed ubiq- uitously in pericarp tissue. To evaluate the biochemical character- istics of the products of the Frk1 and Frk2 genes, each cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line defec- tive in hexose phosphorylation and unable to grow on glucose or fructose (Fru). Both Frk1 and Frk2 proteins expressed in yeast conferred the ability...
Guard cells represent a unique single cell-type system for the study of cellular responses to abi... more Guard cells represent a unique single cell-type system for the study of cellular responses to abiotic and biotic perturbations that affect stomatal movement. Decades of effort through both classical physiological and functional genomics approaches have generated an enormous amount of information on the roles of individual metabolites in stomatal guard cell function and physiology. Recent application of metabolomics methods has produced a substantial amount of new information on metabolome control of stomatal movement. In conjunction with other "omics" approaches, the knowledge-base is growing to reach a systems-level description of this single cell-type. Here we summarize current knowledge of the guard cell metabolome and highlight critical metabolites that bear significant impact on future engineering and breeding efforts to generate plants/crops that are resistant to environmental challenges and produce high yield and quality products for food and energy security.
Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydr... more 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.
Stomata, composed of two guard cells, are the gates whose controlled movement allows the plant to... more 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.
Increased expression of the aquaporin NtAQP1, which is known to function as a plasmalemma channel... more Increased expression of the aquaporin NtAQP1, which is known to function as a plasmalemma channel for CO 2 and water, increases the rate of both photosynthesis and transpiration. In contrast, increased expression of Arabidopsis hexokinase1 (AtHXK1), a dual-function enzyme that mediates sugar sensing, decreases the expression of photosynthetic genes and the rate of transpiration and inhibits growth. Here, we show that AtHXK1 also decreases root and stem hydraulic conductivity and leaf mesophyll CO 2 conductance (g m ). Due to their opposite effects on plant development and physiology, we examined the relationship between NtAQP1 and AtHXK1 at the whole-plant level using transgenic tomato plants expressing both genes simultaneously. NtAQP1 significantly improved growth and increased the transpiration rates of AtHXK1expressing plants. Reciprocal grafting experiments indicated that this complementation occurs when both genes are expressed simultaneously in the shoot. Yet, NtAQP1 had only a marginal effect on the hydraulic conductivity of the doubletransgenic plants, suggesting that the complementary effect of NtAQP1 is unrelated to shoot water transport. Rather, NtAQP1 significantly increased leaf mesophyll CO 2 conductance and enhanced the rate of photosynthesis, suggesting that NtAQP1 facilitated the growth of the double-transgenic plants by enhancing mesophyll conductance of CO 2 .
Two different partial sequences encoding putative hexokinase (HXK, ATP: hexose-6-phosphotransfera... more Two different partial sequences encoding putative hexokinase (HXK, ATP: hexose-6-phosphotransferase, EC 2.7.1.1) were isolated from tomato (Lycopersicon esculentum) by RT-PCR using degenerate primers. Southern blot analysis suggested the existence of two divergent HXK genes. A complete cDNA of one HXK was isolated by screening a cDNA library prepared from young cherry tomato fruit. The 1770 bp cDNA of LeHXK2 contained
The LeFRK2 gene product has been proposed to play a role in transient starch accumulation in deve... more The LeFRK2 gene product has been proposed to play a role in transient starch accumulation in developing tomato (Lycopersicon esculentum) fruits. To analyze the role of LeFRK2 in starch biosynthesis, we produced transgenic tomato plants with sense and antisense expression of StFRK, the potato homologue to LeFRK2. Fruits of homozygous plants expressing sense or antisense StFRK exhibited suppression of LeFRK2, concomitantly with specific elimination of the FKI isozyme, indicating that FKI is the gene product of LeFRK2. The activity of fructokinase was reduced in antisense and sense homozygous fruits and increased in fruits of sense hemizygous plants. The modified activities of fructokinase led to small but significant changes both in the steady state levels of sugars and in the level of phosphorylated sugars in fruits. However, fruits lacking FKI had increased rather than decreased starch content. We therefore concluded that LeFRK2 is not required for transient starch accumulation in tomato fruits. Rather, LeFRK2 might have a role in the regulation of sucrose import into tomato fruits.
A full-length cDNA encoding a novel fourth fructokinase, LeFRK3, was cloned from green tomato (Ly... more A full-length cDNA encoding a novel fourth fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate
The segregation of the nucleolus during mitosis was examined in Saccharomyces cerevisiae and Schi... more The segregation of the nucleolus during mitosis was examined in Saccharomyces cerevisiae and Schizosaccharomyces pombe by indirect immunofluorescence using antibodies directed to highly conserved anti-nucleolus antigens. In mitotic S. pombe cells, the nucleolus appears to trail the bulk of the DNA. In wild-type cells of S. cerevisiae, the nucleolus segregates alongside the bulk of the genomic DNA. Based on its distance from the centromere, we would expect the rDNA in both organisms to segregate behind the majority of the genomic DNA, if telomeric regions trail centromeric regions as in other eukaryotes. We therefore suggest that in S. cerevisiae the nucleolus is attached to other parts of the nucleus which enable it to segregate along with the bulk of the DNA. The segregation of the nucleolus in topoisomerase mutants and nuclear division mutants of S. cerevisiae was also investigated. In cdc14 mutants which arrest at late anaphase, the vast majority of the DNA is separated, but the ...
Carbohydrates are essential for male gametophyte development. However, our understanding of the m... more Carbohydrates are essential for male gametophyte development. However, our understanding of the mechanism by which the sugar supply is controlled in the stamen is still in its infancy. We previously reported on the stamen-specific expression of the tomato (Solanum lycopersicum) sugar metabolic gene, fructokinase 4 (SlFRK4). Here, we present the cloning and the characterization of the SlFRK4 promoter and show its differential activation during anther development. We also show that the tissue-specific expression of SlFRK4 promoter is maintained in Arabidopsis thaliana. By histochemical analyses of the GUS reporter gene and DTA toxin driven by the SlFRK4 promoter, we show that the SlFRK4 promoter is gradually activated in pollen grains throughout the later stages of anther development and upon pollen germination. In addition, we analyzed the expression profile of SlFRK4 and other sugar metabolic genes and found that SlFRK4 and the invertase LIN7 are co-expressed in mature and germinated pollen. These findings point to the existence of a specialized mechanism in which carbohydrates are provided to the male gametophyte during the later stages of its development and suggest a valuable tool for manipulating the development of male gametophytes in crop species.
Increased expression of the aquaporin NtAQP1, which is known to function as a plasmalemma channel... more Increased expression of the aquaporin NtAQP1, which is known to function as a plasmalemma channel for CO 2 and water, increases the rate of both photosynthesis and transpiration. In contrast, increased expression of Arabidopsis hexokinase1 (AtHXK1), a dual-function enzyme that mediates sugar sensing, decreases the expression of photosynthetic genes and the rate of transpiration and inhibits growth. Here, we show that AtHXK1 also decreases root and stem hydraulic conductivity and leaf mesophyll CO 2 conductance (g m ). Due to their opposite effects on plant development and physiology, we examined the relationship between NtAQP1 and AtHXK1 at the whole-plant level using transgenic tomato plants expressing both genes simultaneously. NtAQP1 significantly improved growth and increased the transpiration rates of AtHXK1expressing plants. Reciprocal grafting experiments indicated that this complementation occurs when both genes are expressed simultaneously in the shoot. Yet, NtAQP1 had only a marginal effect on the hydraulic conductivity of the doubletransgenic plants, suggesting that the complementary effect of NtAQP1 is unrelated to shoot water transport. Rather, NtAQP1 significantly increased leaf mesophyll CO 2 conductance and enhanced the rate of photosynthesis, suggesting that NtAQP1 facilitated the growth of the double-transgenic plants by enhancing mesophyll conductance of CO 2 .
Our understanding of the cellular role of aquaporins (AQPs) in the regulation of whole-plant hydr... more 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.
Hexose sugars, such as glucose and fructose produced in plants, are ubiquitous in most organisms ... more Hexose sugars, such as glucose and fructose produced in plants, are ubiquitous in most organisms and are the origin of most of the organic matter found in nature. To be utilized, hexose sugars must first be phosphorylated. The central role of hexose-phosphorylating enzymes has attracted the attention of many researchers, leading to novel discoveries. Only two families of enzymes capable of phosphorylating glucose and fructose have been identified in plants; hexokinases (HXKs), and fructokinases (FRKs). Intensive investigations of these two families in numerous plant species have yielded a wealth of knowledge regarding the genes number, enzymatic characterization, intracellular localization, and developmental and physiological roles of several HXKs and FRKs. The emerging picture indicates that HXK and FRK enzymes found at specific intracellular locations play distinct roles in plant metabolism and development. Individual HXKs were shown for the first time to be dual-function enzymes -sensing sugar levels independent of their catalytic activity and controlling gene expression and major developmental pathways, as well as hormonal interactions. FRK, on the other hand, seems to play a central metabolic role in vascular tissues, controlling the amounts of sugars allocated for vascular development. While a clearer picture of the roles of these two types of enzymes is emerging, many questions remain unsolved, such as the specific tissues and types of cells in which these enzymes function, the roles of individual HXK and FRK genes, and how these enzymes interact with hormones in the regulation of developmental processes. It is anticipated that ongoing efforts will broaden our knowledge of these important plant enzymes and their potential uses in the modification of plant traits.
Hexokinases (HXKs), catalysts of the first essential step in glucose metabolism, have emerged as ... more Hexokinases (HXKs), catalysts of the first essential step in glucose metabolism, have emerged as important enzymes that mediate sugar sensing in many organisms, including plants. The presence of several types of plant HXK isozymes, located in different intracellular locations, has been suggested. However, recent studies have indicated that most plants have only two types of HXKs, a plastidic stromal isozyme and membrane-associated isozymes located mainly adjacent to the mitochondria, but also in the nucleus. The membrane-associated isozymes are involved in sugar sensing and regulate gene expression. The central role of HXKs in plant development and the increasing interest in their role necessitate the correction of inaccuracies that have spread concerning the substrate specificity and intracellular localization of HXK isozymes, as these inaccuracies are affecting the hypothesized roles presented for these isozymes and shaping future research in this active field.
Sarcotoxin IA is a cecropin-type antibacterial protein produced by the flesh fly,Sarcophaga pereg... more Sarcotoxin IA is a cecropin-type antibacterial protein produced by the flesh fly,Sarcophaga peregrina.Similar to other bactericidal small proteins produced by insects, sarcotoxin IA is released into the hemolymph of larvae and nymphs upon mechanical injury or bacterial infection. The gene (sarco) that encodes this toxin was introduced intoSaccharomyces cerevisiaeyeast cells and was expressed under a constitutive yeast promoter. The transformed
By using immunolocalization and differential extraction methods we show that only apoplastic inve... more By using immunolocalization and differential extraction methods we show that only apoplastic invertase, but not vacuolar invertase, was present in the mature, sucrose-accumulating L. hirsutum pericarp. In contrast, in the hexoseaccumulating L. esculentum fruit, both the apoplastic and vacuolar invertase activities and protein content increase in the mature fruit. Quantitative expression studies of the soluble invertase gene (TIV1) and the apoplastic invertase genes (LINs) showed that only TIV1 gene expression could account for the species and developmental differences of both soluble and insoluble enzyme activity of the pericarp. The expression of the LIN genes encoding for apoplastic tomato invertases was unrelated to the differences in bound enzyme activity and
Two divergent genes encoding fructokinase, Frk1 and Frk2, have been previously shown to be expres... more Two divergent genes encoding fructokinase, Frk1 and Frk2, have been previously shown to be expressed in tomato (Lycopersicon esculentum L.) and have now been further characterized with re- gard to their spatial expression and the enzymic properties of the encoded proteins. Frk1 and Frk2 mRNA levels were coordinately induced by exogenous sugar, indicating that both belong to the growing class of sugar-regulated genes. However, in situ hybridiza- tion indicated that Frk1 and Frk2 were expressed in a spatially distinct manner, with Frk2 mRNA primarily localized in cells of the fruit pericarp, which store starch, and Frk1 mRNA distributed ubiq- uitously in pericarp tissue. To evaluate the biochemical character- istics of the products of the Frk1 and Frk2 genes, each cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line defec- tive in hexose phosphorylation and unable to grow on glucose or fructose (Fru). Both Frk1 and Frk2 proteins expressed in yeast conferred the ability...
Uploads
Papers by David Granot