Mohammad-Reza Hajirezaei

RNAi-mediated tocopherol deficiency impairs photoassimilate export in transgenic potato plants.

Tocopherols (vitamin E) are lipophilic antioxidants presumed to play a key role in protecting chloroplast membranes and the photosynthetic apparatus from photooxidative damage. Additional nonantioxidant functions of tocopherols have been proposed after the recent finding that the Suc export defective1 maize (Zea mays) mutant (sxd1) carries a defect in tocopherol cyclase (TC) and thus is devoid of tocopherols. However, the corresponding vitamin E deficient1 Arabidopsis mutant (vte1) lacks a phenotype analogous to sxd1, suggesting differences in tocopherol function between C4 and C3 plants. Therefore, in this study, the potato (Solanum tuberosum) ortholog of SXD1 was isolated and functionally characterized. StSXD1 encoded a protein with high TC activity in vitro, and chloroplastic localization was demonstrated by transient expression of green fluorescent protein-tagged fusion constructs. RNAi-mediated silencing of StSXD1 in transgenic potato plants resulted in the disruption of TC activity and severe tocopherol deficiency similar to the orthologous sxd1 and vte1 mutants. The nearly complete absence of tocopherols caused a characteristic photoassimilate export-defective phenotype comparable to sxd1, which appeared to be a consequence of vascular-specific callose deposition observed in source leaves. CO2 assimilation rates and photosynthetic gene expression were decreased in source leaves in close correlation with excess sugar accumulation, suggesting a carbohydrate-mediated feedback inhibition rather than a direct impact of tocopherol deficiency on photosynthetic capacity. This conclusion is further supported by an increased photosynthetic capacity of young leaves regardless of decreased tocopherol levels. Our data provide evidence that tocopherol deficiency leads to impaired photoassimilate export from source leaves in both monocot and dicot plant species and suggest significant differences among C3 plants in response to tocopherol reduction.

The sucrose transporter StSUT1 localizes to sieve elements in potato tuber phloem and influences tuber physiology and development.

The sucrose (Suc) H+-cotransporterStSUT1 from potato (Solanum tuberosum), which is essential for long-distance transport of Suc and assumed to play a role in phloem loading in mature leaves, was found to be expressed in sink tubers. To answer the question of whether SUT1 serves a function in phloem unloading in tubers, the promoter was fused to gusA and expression was analyzed in transgenic potato. SUT1 expression was unexpectedly detected not in tuber parenchyma but in the phloem of sink tubers. Immunolocalization demonstrated that StSUT1 protein was present only in sieve elements of sink tubers, cells normally involved in export of Suc from the phloem to supply developing tubers, raising the question of the role of SUT1 in tubers. SUT1 expression was inhibited by antisense in transgenic potato plants using a class I patatin promoter B33, which is primarily expressed in the phloem of developing tubers. ReducedSUT1 expression in tubers did not affect aboveground organs but led to reduced fresh weight accumulation during early stages of tuber development, indicating that in this phase SUT1 plays an important role for sugar transport. Changes in Suc- and starch-modifying enzyme activities and metabolite profiles are consistent with the developmental switch in unloading mechanisms. Altogether, the findings may suggest a role of SUT1 in retrieval of Suc from the apoplasm, thereby regulating the osmotic potential in the extracellular space, or a direct role in phloem unloading acting as a phloem exporter transferring Suc from the sieve elements into the apoplasm.

Functional replacement of ferredoxin by a cyanobacterial flavodoxin in tobacco confers broad-range stress tolerance.

Chloroplast ferredoxin (Fd) plays a pivotal role in plant cell metabolism by delivering reducing equivalents to various essential oxidoreductive pathways. Fd levels decrease under adverse environmental conditions in many microorganisms, including cyanobacteria, which share a common ancestor with chloroplasts. Conversely, stress situations induce the synthesis of flavodoxin (Fld), an electron carrier flavoprotein not found in plants, which can efficiently replace Fd in most electron transfer processes. We report here that chloroplast Fd also declined in plants exposed to oxidants or stress conditions. A purified cyanobacterial Fld was able to mediate plant Fd-dependent reactions in vitro, including NADP+ and thioredoxin reduction. Tobacco (Nicotiana tabacum) plants expressing Fld in chloroplasts displayed increased tolerance to multiple sources of stress, including redox-cycling herbicides, extreme temperatures, high irradiation, water deficit, and UV radiation. Oxidant buildup and oxidative inactivation of thioredoxin-dependent plastidic enzymes were decreased in stressed plants expressing plastid-targeted Fld, suggesting that development of the tolerant phenotype relied on productive interaction of this flavoprotein with Fd-dependent oxidoreductive pathways of the host, most remarkably, thioredoxin reduction. The use of Fld provides new tools to investigate the requirements of photosynthesis in planta and to increase plant stress tolerance based on the introduction of a cyanobacterial product that is free from endogenous regulation in higher plants.

A proteomics view on the role of drought-induced senescence and oxidative stress defense in enhanced stem reserves remobilization in wheat

Drought is one of the major factors limiting the yield of wheat (Triticum aestivum L.) particularly during grain filling. Under terminal drought condition, remobilization of pre-stored carbohydrates in wheat stem to grain has a major contribution in yield. To determine the molecular mechanism of stem reserve utilization under drought condition, we compared stem proteome patterns of two contrasting wheat landraces (N49 and N14) under a progressive post-anthesis drought stress, during which period N49 peduncle showed remarkably higher stem reserves remobilization efficiency compared to N14. Out of 830 protein spots reproducibly detected and analyzed on two-dimensional electrophoresis gels, 135 spots showed significant changes in at least one landrace. The highest number of differentially expressed proteins was observed in landrace N49 at 20days after anthesis when active remobilization of dry matter was observed, suggesting a possible involvement of these proteins in effective stem reserve remobilization of N49. The identification of 82 of differentially expressed proteins using mass spectrometry revealed a coordinated expression of proteins involved in leaf senescence, oxidative stress defense, signal transduction, metabolisms and photosynthesis which might enable N49 to efficiently remobilized its stem reserves compared to N14. The up-regulation of several senescence-associated proteins and breakdown of photosynthetic proteins in N49 might reflect the fact that N49 increased carbon remobilization from the stem to the grains by enhancing senescence. Furthermore, the up-regulation of several oxidative stress defense proteins in N49 might suggest a more effective protection against oxidative stress during senescence in order to protect stem cells from premature cell death. Our results suggest that wheat plant might response to soil drying by efficiently remobilize assimilates from stem to grain through coordinated gene expression.

Altering trehalose-6-phosphate content in transgenic potato tubers affects tuber growth and alters responsiveness to hormones during sprouting

Trehalose-6-phosphate (T6P) is a signaling metabolite that regulates carbon metabolism, developmental processes, and growth in plants. In Arabidopsis (Arabidopsis thaliana), T6P signaling is, at least in part, mediated through inhibition of the SNF1-related protein kinase SnRK1. To investigate the role of T6P signaling in a heterotrophic, starch-accumulating storage organ, transgenic potato (Solanum tuberosum) plants with altered T6P levels specifically in their tubers were generated. Transgenic lines with elevated T6P levels (B33-TPS, expressing Escherichia coli osmoregulatory trehalose synthesis A [OtsA], which encodes a T6P synthase) displayed reduced starch content, decreased ATP contents, and increased respiration rate diagnostic for high metabolic activity. On the other hand, lines with significantly reduced T6P (B33-TPP, expressing E. coli OtsB, which encodes a T6P phosphatase) showed accumulation of soluble carbohydrates, hexose phosphates, and ATP, no change in starch when calculated on a fresh weight basis, and a strongly reduced tuber yield. [14C]Glucose feeding to transgenic tubers indicated that carbon partitioning between starch and soluble carbohydrates was not altered. Transcriptional profiling of B33-TPP tubers revealed that target genes of SnRK1 were strongly up-regulated and that T6P inhibited potato tuber SnRK1 activity in vitro. Among the SnRK1 target genes in B33-TPP tubers, those involved in the promotion of cell proliferation and growth were down-regulated, while an inhibitor of cell cycle progression was up-regulated. T6P-accumulating tubers were strongly delayed in sprouting, while those with reduced T6P sprouted earlier than the wild type. Early sprouting of B33-TPP tubers correlated with a reduced abscisic acid content. Collectively, our data indicate that T6P plays an important role for potato tuber growth.

Antisense-inhibition of ADP-glucose pyrophosphorylase in Vicia narbonensis seeds increases soluble sugars and leads to higher water and nitrogen uptake

Abstract. We previously reported on Vicia narbonensis seeds with largely decreased α-D-glucose-1-phosphate adenyltransferase (AGP; EC 2.7.7.27) due to antisense inhibition [H. Weber et al. (2000) Plant J 24:33–43]. In an extended biochemical analysis we show here that in transgenic seeds both AGP activity and ADP-glucose levels were strongly decreased but starch was only moderately reduced and contained less amylose. The flux control coefficient of AGP to starch accumulation was as low as 0.08, i.e. AGP exerts low control on starch biosynthesis in Vicia seeds. Mature cotyledons of antisense seeds had increased contents of lipids, nitrogen and sulfur. The protein content was higher due, in particular, to increased sulfur-rich albumins. Globulin fractions of storage proteins had a lower ratio of legumin to vicilin. Isolated cotyledons partitioned less [14C]sucrose into starch and more into soluble sugars with no change in the protein fraction. Respiration of isolated cotyledons and activities of the major glycolytic and carbohydrate-metabolizing enzymes were not affected. Sucrose and the hexose-phosphate pool were increased but UDP-glucose, 3-phosphoglyceric acid, phosphoenolpyruvate, pyruvate, ATP and ADP were unchanged or even lower, indicating that carbon partitioning changed from starch to sucrose without affecting the glycolytic and respiratory pathways. Soluble compounds were increased but osmolality remained unchanged, indicating compensatory water influx resulting in higher water contents. Developmental patterns of water and nitrogen accumulation suggest a coupled uptake of amino acids and water into cotyledons. We conclude that, due to higher water uptake, transgenic cotyledons take up more amino acids, which become available for protein biosynthesis leading to a higher protein content. Obviously, a substantial part of amino acid uptake into Vicia seeds occurs passively and is osmotically controlled and driven by water influx.

Feedback inhibition of the general phenylpropanoid and flavonol biosynthetic pathways upon a compromised flavonol-3-O-glycosylation

Flavonols, phenylalanine-derived secondary metabolites, have protective and regulatory functions in plants. In Arabidopsis thaliana, they are consecutively glycosylated at their 3-OH and 7-OH groups. UGT78D1 and UGT78D2 are the major flavonol 3-O-glycosyltransferases in Arabidopsis leaves. The ugt78d1 ugt78d2 double mutant, which was strongly compromised in the initial 3-O-glycosylation, showed a severe and specific repression of flavonol biosynthesis, retaining only one-third of the wild-type level. This metabolic phenotype was associated with a repressed transcription of several flavonol biosynthetic genes including the committed step chalcone synthase [(CHS) or TRANSPARENT TESTA 4 (TT4)]. Furthermore, the committed step of the upstream, general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL), was down-regulated in its enzyme activity and in the transcription of the flavonol-related PAL1 and PAL2. However, a complete blocking of flavonoid biosynthesis at CHS released PAL inhibition in a tt4 ugt78d1 ugt78d2 line. PAL activity was even enhanced in the flavonol synthase 1 mutant, which compromises the final formation of flavonol aglycones. The dependence of the PAL feedback inhibition on flavonols was confirmed by chemical complementation of tt4 ugt78d1 ugt78d2 using naringenin, a downstream flavonoid intermediate, which restored the PAL repression. Although aglycones were not analytically detectable, this study provides genetic evidence for a novel, flavonol-dependent feedback inhibition of the flavonol biosynthetic pathway and PAL. It was conditioned by the compromised flavonol-3-O-conjugation and a decrease in flavonol content, yet dependent on a residual, flavonol synthase 1 (FLS1)-related capacity to form flavonol aglycones. Thus, this regulation would not react to a reduced metabolic flux into flavonol biosynthesis, but it might prevent the accumulation of non-glycosylated, toxic flavonols.

Overriding the co-limiting import of carbon and energy into tuber amyloplasts increases the starch content and yield of transgenic potato plants

Transgenic potato (Solanum tuberosum) plants simultaneously over-expressing a pea (Pisum sativum) glucose-6-phosphate/phosphate translocator (GPT) and an Arabidopsis thaliana adenylate translocator (NTT1) in tubers were generated. Double transformants exhibited an enhanced tuber yield of up to 19%, concomitant with an additional increased starch content of up to 28%, compared with control plants. The total starch content produced in tubers per plant was calculated to be increased by up to 44% in double transformants relative to the wild-type. Single over-expression of either gene had no effect on tuber starch content or tuber yield, suggesting that starch formation within amyloplasts is co-limited by the import of energy and the supply of carbon skeletons. As total adenosine diphosphate-glucose pyrophosphorylase and starch synthase activities remained unchanged in double transformants relative to the wild-type, they cannot account for the increased starch content found in tubers of double transformants. Rather, an optimized supply of amyloplasts with adenosine triphosphate and glucose-6-phosphate seems to favour increased starch synthesis, resulting in plants with increased starch content and yield of tubers.

A comparative proteome approach to decipher the mechanism of rice adaptation to phosphorous deficiency.

Mineral deficiency limits crop production in most soils and in Asia alone, about 50% of rice lands are phosphorous deficient. In an attempt to determine the mechanism of rice adaptation to phosphorous deficiency, changes in proteome patterns associated with phosphorous deficiency have been investigated. We analyzed the parental line Nipponbare in comparison to its near isogenic line (NIL6‐4) carrying a major phosphorous uptake QTL (Pup1) on chromosome 12. Using 2‐DE, the proteome pattern of roots grown under 1 and 100 μM phosphorous were compared. Out of 669 proteins reproducibly detected on root 2‐DE gels, 32 proteins showed significant changes in the two genotypes. Of them, 17 proteins showed different responses in two genotypes under stress condition. MS resulted in identification of 26 proteins involved in major phosphorous deficiency adaptation pathways including reactive oxygen scavenging, citric acid cycle, signal transduction, and plant defense responses as well as proteins with unknown function. Our results highlighted a coordinated response in NIL in response to phosphorous deficiency which may confer higher adaptation to nutrient deficiency.

Enhanced plant tolerance to iron starvation by functional substitution of chloroplast ferredoxin with a bacterial flavodoxin

Iron limitation affects one-third of the cultivable land on Earth and represents a major concern for agriculture. It causes decline of many photosynthetic components, including the Fe-S protein ferredoxin (Fd), involved in essential oxidoreductive pathways of chloroplasts. In cyanobacteria and some algae, Fd down-regulation under Fe deficit is compensated by induction of an isofunctional electron carrier, flavodoxin (Fld), a flavin mononucleotide-containing protein not found in plants. Transgenic tobacco lines expressing a cyanobacterial Fld in chloroplasts were able to grow in Fe-deficient media that severely compromised survival of WT plants. Fld expression did not improve Fe uptake or mobilization, and stressed transformants elicited a normal deficit response, including induction of ferric-chelate reductase and metal transporters. However, the presence of Fld did prevent decrease of several photosynthetic proteins (but not Fd) and partially protected photosynthesis from inactivation. It also preserved the activation state of enzymes depending on the Fd-thioredoxin pathway, which correlated with higher levels of intermediates of carbohydrate metabolism and the Calvin cycle, as well as increased contents of sucrose, glutamate, and other amino acids. These metabolic routes depend, directly or indirectly, on the provision of reduced Fd. The results indicate that Fld could compensate Fd decline during episodes of Fe deficiency by productively interacting with Fd-dependent pathways of the host, providing fresh genetic resources for the design of plants able to survive in Fe-poor lands.