Arabidopsis disrupted in SQD2 encoding sulfolipid synthase is impaired in phosphate-limited growth

The sulfolipid sulfoquinovosyldiacylglycerol is one of the three nonphosphorous glycolipids that provide the bulk of the structural lipids in photosynthetic membranes of seed plants. Unlike the galactolipids, sulfolipid is anionic at physiological pH because of its 6-deoxy-6-sulfonate-glucose (sulfoquinovose) head group. The biosynthesis of this lipid proceeds in two steps: first, the assembly of UDP-sulfoquinovose from UDP-glucose and sulfite, and second, the transfer of the sulfoquinovose moiety from UDP-sulfoquinovose to diacylglycerol. The first reaction is catalyzed by the SQD1 protein in Arabidopsis. Here we describe the identification of the SQD2 gene of Arabidopsis. We propose that this gene encodes the sulfoquinovosyltransferase catalyzing the second step of sulfolipid biosynthesis. Expression of SQD1 and SQD2 in Escherichia coli reconstituted plant sulfolipid biosynthesis in this bacterium. Insertion of a transfer DNA into this gene in Arabidopsis led to complete lack of sulfolipid in the respective sqd2 mutant. This mutant showed reduced growth under phosphate-limited growth conditions. The results support the hypothesis that sulfolipid can function as a substitute of anionic phospholipids under phosphate-limited growth conditions. Along with phosphatidylglycerol, sulfolipid contributes to maintaining a negatively charged lipid-water interface, which presumably is required for proper function of photosynthetic membranes.     

The sulfolipid sulfoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances growth under phosphate limitation.

All photosynthetic organisms, with the exception of several species of photosynthetic bacteria, are thought to contain the sulfolipid 6-sulfo-alpha-D-quinovosyldiacylglycerol. The association of this lipid with photosynthetic membranes has led to the assumption that it plays some role in photosynthesis. Stable null mutants of the photosynthetic bacterium Rhodobacter sphaeroides completely lacking sulfolipid were obtained by disruption of the sqdB gene. The ratios of the various components of the photosynthetic electron transport chain, as well as the electron transfer rates during cyclic electron transport, were not altered in the mutants, when grown under optimal conditions. Growth rates of wild type and mutants were identical under a variety of growth conditions, with the exception of phosphate limitation, which resulted in reduced growth of the mutants. Phosphate limitation of the wild type caused a significant reduction in the amount of all phospholipids and an increased amount of sulfolipid. By contrast, the sulfolipid-deficient mutant had reduced levels of phosphatidylcholine and phosphatidylethanolamine but maintained a normal level of phosphatidylglycerol. In addition, two unidentified lipids lacking phosphorus accumulated in the membranes of both wild-type and mutant strains under phosphate limitation. We conclude that sulfolipid plays no significant unique role in photoheterotrophic growth or photosynthetic electron transport in R. sphaeroides but may function as a surrogate for phospholipids, particularly phosphatidylglycerol, under phosphate-limiting conditions.     

Unsaturation of the membrane lipids of chloroplasts stabilizes the photosynthetic machinery against low-temperature photoinhibition in transgenic tobacco plants.

Using tobacco plants that had been transformed with the cDNA for glycerol-3-phosphate acyltransferase, we have demonstrated that chilling tolerance is affected by the levels of unsaturated membrane lipids. In the present study, we examined the effects of the transformation of tobacco plants with cDNA for glycerol-3-phosphate acyltransferase from squash on the unsaturation of fatty acids in thylakoid membrane lipids and the response of photosynthesis to various temperatures. Of the four major lipid classes isolated from the thylakoid membranes, phosphatidylglycerol showed the most conspicuous decrease in the level of unsaturation in the transformed plants. The isolated thylakoid membranes from wild-type and transgenic plants did not significantly differ from each other in terms of the sensitivity of photosystem II to high and low temperatures and also to photoinhibition. However, leaves of the transformed plants were more sensitive to photoinhibition than those of wild-type plants. Moreover, the recovery of photosynthesis from photoinhibition in leaves of wild-type plants was faster than that in leaves of the transgenic tobacco plants. These results suggest that unsaturation of fatty acids of phosphatidylglycerol in thylakoid membranes stabilizes the photosynthetic machinery against low-temperature photoinhibition by accelerating the recovery of the photosystem II protein complex.     

Arabidopsis CHL27, located in both envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide

CHL27, the Arabidopsis homologue to Chlamydomonas Crd1, a plastid-localized putative diiron protein, is required for the synthesis of protochlorophyllide and therefore is a candidate subunit of the aerobic cyclase in chlorophyll biosynthesis. δ-Aminolevulinic acid-fed antisense Arabidopsis plants with reduced amounts of Crd1/CHL27 accumulate Mg-protoporphyrin IX monomethyl ester, the substrate of the cyclase reaction. Mutant plants have chlorotic leaves with reduced abundance of all chlorophyll proteins. Fractionation of Arabidopsis chloroplast membranes shows that Crd1/CHL27 is equally distributed on a membrane-weight basis in the thylakoid and inner-envelope membranes.     

ER-resident proteins PDR2 and LPR1 mediate the developmental response of root meristems to phosphate availability

Inadequate availability of inorganic phosphate (Pi) in the rhizosphere is a common challenge to plants, which activate metabolic and developmental responses to maximize Pi acquisition. The sensory mechanisms that monitor environmental Pi status and regulate root growth via altered meristem activity are unknown. Here, we show that phosphate deficiency response 2 (PDR2) encodes the single P₅-type ATPase of Arabidopsis thaliana. PDR2 functions in the endoplasmic reticulum (ER) and is required for proper expression of scarecrow (SCR), a key regulator of root patterning, and for stem-cell maintenance in Pi-deprived roots. We further show that the multicopper oxidase encoded by low phosphate root 1 (LPR1) is targeted to the ER and that LPR1 and PDR2 interact genetically. Because the expression domains of both genes overlap in the stem-cell niche and distal root meristem, we propose that PDR2 and LPR1 function together in an ER-resident pathway that adjusts root meristem activity to external Pi. Our data indicate that the Pi-conditional root phenotype of pdr2 is not caused by increased Fe availability in low Pi; however, Fe homeostasis modifies the developmental response of root meristems to Pi availability.     

Arabidopsis lipins mediate eukaryotic pathway of lipid metabolism and cope critically with phosphate starvation

Phosphate is an essential nutrient for plant viability. It is well-established that phosphate starvation triggers membrane lipid remodeling, a process that converts significant portion of phospholipids to non-phosphorus-containing galactolipids. This remodeling is mediated by either phospholipase C (PLC) or phospholipase D (PLD) in combination with phosphatidate phosphatase (PAP). Two PLC genes, NPC4 and NPC5, and PLD genes, PLDζ1 and PLDζ2, are shown to be involved in the remodeling. However, gene knockout studies show that none of them plays decisive roles in the remodeling. Thus, although this phenomenon is widely observed among plants, the key enzyme(s) responsible for the lipid remodeling in a whole plant body is unknown; therefore, the physiological significance of this conversion process has remained to be elucidated. We herein focused on PAP as a key enzyme for this adaptation, and identified Arabidopsis lipin homologs, AtPAH1 and AtPAH2, that encode the PAPs involved in galactolipid biosynthesis. Double mutant pah1pah2 plants had decreased phosphatidic acid hydrolysis, thus affecting the eukaryotic pathway of galactolipid synthesis. Upon phosphate starvation, pah1pah2 plants were severely impaired in growth and membrane lipid remodeling. These results indicate that PAH1 and PAH2 are the PAP responsible for the eukaryotic pathway of galactolipid synthesis, and the membrane lipid remodeling mediated by these two enzymes is an essential adaptation mechanism to cope with phosphate starvation.     

High temperature promotes auxin-mediated hypocotyl elongation in Arabidopsis

Physiological studies with excised stem segments have implicated the plant hormone indole-3-acetic acid (IAA or auxin) in the regulation of cell elongation. Supporting evidence from intact plants has been somewhat more difficult to obtain, however. Here, we report the identification and characterization of an auxin-mediated cell elongation growth response in Arabidopsis thaliana. When grown in the light at high temperature (29°C), Arabidopsis seedlings exhibit dramatic hypocotyl elongation compared with seedlings grown at 20°C. This temperature-dependent growth response is sharply reduced by mutations in the auxin response or transport pathways and in seedlings containing reduced levels of free IAA. In contrast, mutants deficient in gibberellin and abscisic acid biosynthesis or in ethylene response are unaffected. Furthermore, we detect a corresponding increase in the level of free IAA in seedlings grown at high temperature, suggesting that temperature regulates auxin synthesis or catabolism to mediate this growth response. Consistent with this possibility, high temperature also stimulates other auxin-mediated processes including auxin-inducible gene expression. Based on these results, we propose that growth at high temperature promotes an increase in auxin levels resulting in increased hypocotyl elongation. These results strongly support the contention that endogenous auxin promotes cell elongation in intact plants.     

Disruption of the Arabidopsis CGI-58 homologue produces Chanarin–Dorfman-like lipid droplet accumulation in plants

CGI-58 is the defective gene in the human neutral lipid storage disease called Chanarin-Dorfman syndrome. This disorder causes intracellular lipid droplets to accumulate in nonadipose tissues, such as skin and blood cells. Here, disruption of the homologous CGI-58 gene in Arabidopsis thaliana resulted in the accumulation of neutral lipid droplets in mature leaves. Mass spectroscopy of isolated lipid droplets from cgi-58 loss-of-function mutants showed they contain triacylglycerols with common leaf-specific fatty acids. Leaves of mature cgi-58 plants exhibited a marked increase in absolute triacylglycerol levels, more than 10-fold higher than in wild-type plants. Lipid levels in the oil-storing seeds of cgi-58 loss-of-function plants were unchanged, and unlike mutations in β-oxidation, the cgi-58 seeds germinated and grew normally, requiring no rescue with sucrose. We conclude that the participation of CGI-58 in neutral lipid homeostasis of nonfat-storing tissues is similar, although not identical, between plant and animal species. This unique insight may have implications for designing a new generation of technologies that enhance the neutral lipid content and composition of crop plants.     

Molecular Identification of Prune Dwarf Virus (PDV) Infecting Sweet Cherry in Canada and Development of a PDV Full-Length Infectious cDNA Clone

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.     

Knock-out mutants from an En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes

A collection of 8,000 Arabidopsis thaliana plants carrying 48,000 insertions of the maize transposable element En-1 has been generated. This population was used for reverse genetic analyses to identify insertions in individual gene loci. By using a PCR-based screening protocol, insertions were found in 55 genes. En-1 showed no preference for transcribed or untranscribed regions nor for a particular orientation relative to the gene of interest. In several cases, En-1 was inserted within a few kilobases upstream or downstream of the gene. En-1 was mobilized from such positions into the respective gene to cause gene disruption. Knock-out alleles of genes involved in flavonoid biosynthesis were generated. One mutant line contained an En-1 insertion in the flavonol synthase gene (FLS) and showed drastically reduced levels of kaempferol. Allelism tests with other lines containing En-1 insertions in the flavanone 3-hydroxylase gene (F3H) demonstrated that TRANSPARENT TESTA 6 (TT6) encodes flavanone 3-hydroxylase. The f3h and fls null mutants complete the set of A. thaliana lines defective in early steps of the flavonoid pathway. These experiments demonstrate the efficiency of the screening method and gene disruption strategy used for assigning functions to genes defined only by sequence.     

Sinorhizobium meliloti phospholipase C required for lipid remodeling during phosphorus limitation

Rhizobia are Gram-negative soil bacteria able to establish nitrogen-fixing root nodules with their respective legume host plants. Besides phosphatidylglycerol, cardiolipin, and phosphatidylethanolamine, rhizobial membranes contain phosphatidylcholine (PC) as a major membrane lipid. Under phosphate-limiting conditions of growth, some bacteria replace their membrane phospholipids with lipids lacking phosphorus. In Sinorhizobium meliloti, these phosphorus-free lipids are sulfoquinovosyl diacylglycerol, ornithine-containing lipid, and diacylglyceryl trimethylhomoserine (DGTS). Pulse–chase experiments suggest that the zwitterionic phospholipids phosphatidylethanolamine and PC act as biosynthetic precursors of DGTS under phosphorus-limiting conditions. A S. meliloti mutant, deficient in the predicted phosphatase {"type":"entrez-protein","attrs":{"text":"SMc00171","term_id":"1174089800","term_text":"SMC00171"}}SMc00171 was unable to degrade PC or to form DGTS in a similar way as the wild type. Cell-free extracts of Escherichia coli, in which {"type":"entrez-protein","attrs":{"text":"SMc00171","term_id":"1174089800","term_text":"SMC00171"}}SMc00171 had been expressed, convert PC to phosphocholine and diacylglycerol, showing that {"type":"entrez-protein","attrs":{"text":"SMc00171","term_id":"1174089800","term_text":"SMC00171"}}SMc00171 functions as a phospholipase C. Diacylglycerol , in turn, is the lipid anchor from which biosynthesis is initiated during the formation of the phosphorus-free membrane lipid DGTS. Inorganic phosphate can be liberated from phosphocholine. These data suggest that, in S. meliloti under phosphate-limiting conditions, membrane phospholipids provide a pool for metabolizable inorganic phosphate, which can be used for the synthesis of other essential phosphorus-containing biomolecules. This is an example of an intracellular phospholipase C in a bacterial system; however, the ability to degrade endogenous preexisting membrane phospholipids as a source of phosphorus may be a general property of Gram-negative soil bacteria.     

The tomato DWARF enzyme catalyses C-6 oxidation in brassinosteroid biosynthesis

Brassinosteroids (BRs) are steroidal plant hormones essential for normal plant growth and development. Mutants in the biosynthesis or perception of BRs are usually dwarf. The tomato Dwarf gene (D), which was predicted to encode a cytochrome P450 enzyme (P450) with homology to other P450s involved in BR biosynthesis, was cloned previously. Here, we show that DWARF catalyses the C-6 oxidation of 6-deoxocastasterone (6-deoxoCS) to castasterone (CS), the immediate precursor of brassinolide. To do this, we first confirmed that the D cDNA complemented the mutant light- and dark-grown phenotypes of the extreme dwarf (d^x) allele of tomato. To identify a substrate for the DWARF enzyme, exogenous application of BR intermediates to d^x plants was carried out. C-6 oxoBR intermediates enhanced hypocotyl elongation whereas the C-6 deoxoBR, 6-deoxoCS, had little effect. Quantitative analysis of endogenous BR levels in tomato showed mainly the presence of 6-deoxoBRs. Furthermore, d^x plants were found to lack CS and had a high level of 6-deoxoCS in comparison to D plants that had CS and a lower level of 6-deoxoCS. Confirmation that DWARF catalyzed the C-6 oxidation of 6-deoxoCS to CS was obtained by functional expression of DWARF in yeast. In these experiments, the intermediate 6α-hydroxycastasterone was identified, indicating that DWARF catalyzes two steps in BR biosynthesis. These data show that DWARF is involved in the C-6 oxidation in BR biosynthesis.     

SAR11 lipid renovation in response to phosphate starvation

Phytoplankton inhabiting oligotrophic ocean gyres actively reduce their phosphorus demand by replacing polar membrane phospholipids with those lacking phosphorus. Although the synthesis of nonphosphorus lipids is well documented in some heterotrophic bacterial lineages, phosphorus-free lipid synthesis in oligotrophic marine chemoheterotrophs has not been directly demonstrated, implying they are disadvantaged in phosphate-deplete ecosystems, relative to phytoplankton. Here, we show the SAR11 clade chemoheterotroph Pelagibacter sp. str. HTCC7211 renovates membrane lipids when phosphate starved by replacing a portion of its phospholipids with monoglucosyl- and glucuronosyl-diacylglycerols and by synthesizing new ornithine lipids. Lipid profiles of cells grown with excess phosphate consisted entirely of phospholipids. Conversely, up to 40% of the total lipids were converted to nonphosphorus lipids when cells were starved for phosphate, or when growing on methylphosphonate. Cells sequentially limited by phosphate and methylphosphonate transformed >75% of their lipids to phosphorus-free analogs. During phosphate starvation, a four-gene cluster was significantly up-regulated that likely encodes the enzymes responsible for lipid renovation. These genes were found in Pelagibacterales strains isolated from a phosphate-deficient ocean gyre, but not in other strains from coastal environments, suggesting alternate lipid synthesis is a specific adaptation to phosphate scarcity. Similar gene clusters are found in the genomes of other marine α-proteobacteria, implying lipid renovation is a common strategy used by heterotrophic cells to reduce their requirement for phosphorus in oligotrophic habitats.Microbes primarily assimilate phosphorus (P) in its +5 valence state (phosphate; P_i), which comprises ∼3% of total cellular mass as a structural constituent of nucleic acids and phospholipids, and is intimately involved in energy metabolism and some transport functions (via ATP hydrolysis) (1). In oligotrophic ocean gyres, P_i concentrations are extremely low (0.2–1.0 nM in the Sargasso Sea; ref. 2) and the availability of P_i can limit bacterial and primary production (2–5). Microbes inhabiting these low P_i environments have evolved numerous strategies to maintain growth and enhance their competitiveness for trace amounts of P_i. These mechanisms are commonly induced by P_i starvation and include one or more of the following: (i) expression of high affinity P_i transporters (6); (ii) reduction of cellular P_i quotas (7, 8); (iii) utilization of alternate phosphorus sources (9, 10); and (iv) polyphosphate storage and breakdown (11, 12). Such strategies facilitate survival in the face of P_i insufficiency.Polar membrane lipids are a substantial cellular sink for phosphate in bacteria. Structural lipids consist of glycerol esterified to hydrophobic fatty acid chains (diacylglycerol) and a hydrophilic polar head group, which commonly contains P_i (phospholipids). When grown with sufficient P_i, phospholipids in Gram-negative marine bacteria frequently include phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and diphosphatidylglycerol (13). However, when P_i is low or limiting, many microbes replace phospholipids with those that lack phosphorus (14–16). Nonphosphorus polar head groups are structurally diverse and include sulfoquinovose (sulfolipids) (17), various monosaccharides and disaccharides (glycolipids; reviewed in ref. 18), ornithine, or other amino acids (reviewed in ref. 19). When P_i stressed, the marine cyanobacteria Prochlorococcus and Synechococcus reduce their P_i demand by 0.5–8.6 attomoles P per cell by substituting phosphorus-containing lipids with sulfolipids; depending on the strain, this reduction equates to 10–86% of the P bound in their nucleic acids (7). There is indirect evidence natural populations of marine chemoheterotrophic bacteria also use nonphosphorus lipids in response to P_i deprivation. Bacterioplankton collected in the Sargasso Sea had greater concentrations of nonphosphorus lipids than those from adjacent regions of the North Atlantic where P_i was relatively abundant (20).Oligotrophic bacteria belonging to the SAR11 clade (Pelagibacterales) of α-proteobacteria are numerically dominant chemoheterotrophs in marine euphotic zones worldwide (21). Pelagibacterales cells are small (volume of 0.01 μm³; ref. 22) and contain streamlined genomes (23, 24). The reduced cell and genome size likely stem from natural selection to reduce the overhead cost of replication in oligotrophic ocean gyres where P and N may periodically limit growth (25, 26). Despite their abundance in low P_i environments, it remains unknown whether Pelagibacterales synthesize phosphorus-free lipids to reduce their P quota. The genome of Pelagibacter ubique str. HTCC1062 lacks genes predicted to encode proteins used in sulfolipid, betaine, or ornithine lipid biosynthesis, suggesting this strain is unable to modulate lipid composition in response to P_i availability (7, 8). Previous laboratory experiments partially supported this prediction by showing that P. ubique lacks nonphosphorus lipids when grown under P_i replete conditions; however, lipids from P_i limited cells were not examined in that research (7). Relative to P. ubique, a Sargasso Sea isolate, Pelagibacter sp. str. HTCC7211 (str. HTCC7211) contains extended genetic inventory associated with P_i acquisition, storage, and metabolism (10). When P_i limited, str. HTCC7211 induces a suite of these genes, including both inorganic (pstSCAB) and organophosphate (phnCDEE₂) ABC transporters and the C–P lyase complex (phnGHIJKLNM), required for phosphonate degradation (10). Laboratory experiments have distinctly linked the expression of these genes to the utilization of both phosphate esters and phosphonates (including methylphosphonate; MPn) (10). This finding indicates organophosphate utilization is one strategy str. HTCC7211 employs to evade P_i growth limitation.While examining gene expression profiles of P_i starved str. HTCC7211 cultures, we observed the unexpected up-regulation of a four gene cluster proximal to the collection of P uptake genes on the str. HTCC7211 chromosome. Two of the genes were annotated as “putative hemolysins,” one as a “glycosyltransferase,” and a “metallophosphatase.” Comparative genomic examination of these genes led us to hypothesize that the four genes might be involved in the restructuring of lipid polar head groups and the synthesis of nonphosphorus lipids. Herein, we present the results of laboratory experiments designed to test the potential for synthesis of nonphosphorus lipids in response to P_i stress by Pelagibacter.     

Cloned DNA sequences complementary to mRNAs encoding precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase and a chlorophyll a/b binding polypeptide

Double-stranded cDNA was synthesized from pea poly(A)-containing mRNA and inserted into the Pst I site of the bacterial plasmid pBR322 by the addition of synthetic oligonucleotide linkers. Bacterial colonies containing recombinant plasmids were detected by hybridization to partially purified mRNAs and further characterized by cell-free translation of hybridization-selected mRNAs. To confirm the identity of cDNA clones encoding chloroplast polypeptides, we incubated translation products derived from complementary mRNAs with intact chloroplasts in vitro. After uptake, precursor polypeptides were converted to their mature size and identified by fractionation of the chloroplast stroma and thylakoid membranes. By using these procedures, we have isolated and characterized cDNA clones encoding the two major cytoplasmically synthesized chloroplast proteins: the small subunit of ribulose-1,5-bisphosphate carboxylase and a constituent polypeptide (polypeptide 15) of the light-harvesting chlorophyll a/b-protein complex. Similarly, a third cDNA clone was isolated and shown to encode a 22,000-dalton thylakoid membrane polypeptide.     

Extracellular transmission of a DNA mycovirus and its use as a natural fungicide

Mycoviruses are thought not to be infectious as free particles and to lack an extracellular phase in their life cycles, limiting the broad use of hypovirulence-associated mycoviruses in controlling fungal disease. Here, we demonstrate that purified particles of a DNA mycovirus, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), are infectious when applied extracellularly to its host Sclerotinia sclerotiorum. Virus particles isolated from an infected host can infect the hyphae of virus-free S. sclerotiorum directly when applied to hyphae grown on potato dextrose agar or sprayed on leaves of Arabidopsis thaliana and Brassica napus, regardless of vegetative compatibility affiliation. When applied to leaves, the virus can suppress the development of lesions. SsHADV-1 can also reduce disease severity and enhance rapeseed yield significantly under field conditions. SsHADV-1 has a narrow host range; it can infect Sclerotinia minor and Sclerotinia nivalis, sister species of S. sclerotiorum, and cause debilitation of these two fungi, but cannot infect or transfect other tested fungi, such as Botrytis cinerea, which shares the same family with S. sclerotiorum. Virus particles are likely to be very stable on the leaves of A. thaliana plants because viral DNA could be detected at 15 d postinoculation on unwounded leaves and at 10 d postinoculation on wounded leaves, respectively; however, this virus could not infect and move in plant cells. Our findings may prompt a reconsideration of the generalization that mycoviruses lack an extracellular phase in their life cycles and stimulate the search for other DNA mycoviruses with potential use as natural fungicides.     

Adaptation of the symbiotic Mesorhizobium–chickpea relationship to phosphate deficiency relies on reprogramming of whole-plant metabolism

Low inorganic phosphate (Pi) availability is a major constraint for efficient nitrogen fixation in legumes, including chickpea. To elucidate the mechanisms involved in nodule acclimation to low Pi availability, two Mesorhizobium–chickpea associations exhibiting differential symbiotic performances, Mesorhizobium ciceri CP-31 (McCP-31)–chickpea and Mesorhizobium mediterranum SWRI9 (MmSWRI9)–chickpea, were comprehensively studied under both control and low Pi conditions. MmSWRI9–chickpea showed a lower symbiotic efficiency under low Pi availability than McCP-31–chickpea as evidenced by reduced growth parameters and down-regulation of nifD and nifK. These differences can be attributed to decline in Pi level in MmSWRI9-induced nodules under low Pi stress, which coincided with up-regulation of several key Pi starvation-responsive genes, and accumulation of asparagine in nodules and the levels of identified amino acids in Pi-deficient leaves of MmSWRI9-inoculated plants exceeding the shoot nitrogen requirement during Pi starvation, indicative of nitrogen feedback inhibition. Conversely, Pi levels increased in nodules of Pi-stressed McCP-31–inoculated plants, because these plants evolved various metabolic and biochemical strategies to maintain nodular Pi homeostasis under Pi deficiency. These adaptations involve the activation of alternative pathways of carbon metabolism, enhanced production and exudation of organic acids from roots into the rhizosphere, and the ability to protect nodule metabolism against Pi deficiency-induced oxidative stress. Collectively, the adaptation of symbiotic efficiency under Pi deficiency resulted from highly coordinated processes with an extensive reprogramming of whole-plant metabolism. The findings of this study will enable us to design effective breeding and genetic engineering strategies to enhance symbiotic efficiency in legume crops.Phosphorus plays a critical role in numerous plant metabolic processes and contributes to the biosynthesis of cellular macromolecules, such as ATP, nucleic acids, phospholipids, and phosphorylated sugars (1). Thus, phosphorus has been established as one of the most important elements required for normal plant growth and development (1). Unfortunately, limited availability of inorganic phosphate (Pi), which is the only absorbable form of phosphorus for plants, in soils is nearly universal, because Pi readily forms various insoluble compounds with metals, such as calcium and iron in alkaline and acidic soils, respectively (1, 2). Pi deficiency can be overcome by the application of Pi fertilizers; however, the excessive use of chemical fertilizers can have serious environmental consequences, including the contamination of soil and water resources (1). Additionally, the global demand for and use of Pi fertilizers are projected to increase significantly with the explosive growth of the global population. Thus, it has been predicted that global Pi reserves will be depleted within 100 y or even sooner (3).Symbiotic nitrogen fixation (SNF) in dinitrogen (N₂)-fixing legumes is dramatically affected by numerous environmental limitations (4). Among the stressors, low Pi availability is a major restriction of SNF capacity, because this process depends on a series of energy-demanding metabolic steps. Reductions in the concentration of ATP and energy charge in Pi-deficient nodules result in significant declines in nitrogenase activity, SNF capacity, and ultimately, the growth and productivity of legume crops (5). Thus, an improvement in Pi levels in the nodules of plants grown in soils with low Pi availability can contribute to a more efficient atmospheric N₂ fixing capacity and therefore, greater productivity of legumes (6). Although low Pi availability has been well-documented as a major restriction for SNF capacity, which imposes serious limitations on legume growth and crop production, little information is available pertaining to the mechanisms responsible for the decrease in SNF capacity under Pi starvation (7). A deep understanding of the complex strategies by which legume metabolism deals with nutritional Pi deficiency can pave the way to breeding crop plants with enhanced symbiotic efficiency or developing them through biotechnological strategies. The development of Pi-efficient crop plants that can grow and yield better under low Pi supply would have a significant beneficial impact on agricultural sustainability (8).Being one of the major pulse crops in the world, chickpea (Cicer arietinum L.) is traditionally cultivated in many countries of different continents (9). It ranks third globally in total yield among grain legume crops (10). Chickpea has a high nutritive value and serves as an important source of protein in developing countries; also, it has the ability to increase soil fertility in terms of soil nitrogen (N) content because of the activity of SNF (11). Among legumes, chickpea has a superior ability to fix atmospheric N₂ through its symbiotic relationship with compatible and effective Rhizobium strains and therefore, can reduce the need for chemical fertilizers, thus minimizing the adverse environmental effects of synthetic fertilizers. However, low soil fertility, particularly Pi deficiency, imposes an important constraint on chickpea crop production, especially in tropical and subtropical areas of Africa and Asia (12). Thus, the selection of Rhizobium–chickpea symbiotic associations with efficient N₂ fixing capability under low Pi availability can help improve chickpea production in low Pi fields.In this study, two Mesorhizobium strains inoculated on chickpea cultivar ILC482 were initially found to have differential symbiotic performances under both control and low Pi stress conditions. A comprehensive examination of the metabolic, biochemical, and molecular mechanisms underlying the differential SNF responses to Pi deficiency in these two Mesorhizobium–chickpea symbiotic associations allowed the identification of the key factors modulating SNF capacity in chickpea under Pi starvation and provided evidence that changes in the metabolism in plant organs other than nodules play an important role in determining the SNF capacity of chickpea plants.     

Gene-for-gene disease resistance without the hypersensitive response in Arabidopsis dnd1 mutant

The cell death response known as the hypersensitive response (HR) is a central feature of gene-for-gene plant disease resistance. A mutant line of Arabidopsis thaliana was identified in which effective gene-for-gene resistance occurs despite the virtual absence of HR cell death. Plants mutated at the DND1 locus are defective in HR cell death but retain characteristic responses to avirulent Pseudomonas syringae such as induction of pathogenesis-related gene expression and strong restriction of pathogen growth. Mutant dnd1 plants also exhibit enhanced resistance against a broad spectrum of virulent fungal, bacterial, and viral pathogens. The resistance against virulent pathogens in dnd1 plants is quantitatively less strong and is differentiable from the gene-for-gene resistance mediated by resistance genes RPS2 and RPM1. Levels of salicylic acid compounds and mRNAs for pathogenesis-related genes are elevated constitutively in dnd1 plants. This constitutive induction of systemic acquired resistance may substitute for HR cell death in potentiating the stronger gene-for-gene defense response. Although cell death may contribute to defense signal transduction in wild-type plants, the dnd1 mutant demonstrates that strong restriction of pathogen growth can occur in the absence of extensive HR cell death in the gene-for-gene resistance response of Arabidopsis against P. syringae.     

The PPH1 phosphatase is specifically involved in LHCII dephosphorylation and state transitions in Arabidopsis

The ability of plants to adapt to changing light conditions depends on a protein kinase network in the chloroplast that leads to the reversible phosphorylation of key proteins in the photosynthetic membrane. Phosphorylation regulates, in a process called state transition, a profound reorganization of the electron transfer chain and remodeling of the thylakoid membranes. Phosphorylation governs the association of the mobile part of the light-harvesting antenna LHCII with either photosystem I or photosystem II. Recent work has identified the redox-regulated protein kinase STN7 as a major actor in state transitions, but the nature of the corresponding phosphatases remained unknown. Here we identify a phosphatase of Arabidopsis thaliana, called PPH1, which is specifically required for the dephosphorylation of light-harvesting complex II (LHCII). We show that this single phosphatase is largely responsible for the dephosphorylation of Lhcb1 and Lhcb2 but not of the photosystem II core proteins. PPH1, which belongs to the family of monomeric PP2C type phosphatases, is a chloroplast protein and is mainly associated with the stroma lamellae of the thylakoid membranes. We demonstrate that loss of PPH1 leads to an increase in the antenna size of photosystem I and to a strong impairment of state transitions. Thus phosphorylation and dephosphorylation of LHCII appear to be specifically mediated by the kinase/phosphatase pair STN7 and PPH1. These two proteins emerge as key players in the adaptation of the photosynthetic apparatus to changes in light quality and quantity.