Hari B. Krishnan

Immunochemical studies on the role of the Golgi complex in protein-body formation in rice seeds

Antibodies raised against purified glutelins and prolamines were employed as probes to study the cellular routes by which these proteins are deposited into protein bodies of rice (Oryza sativa L.) endosperm. Three morphologically distinct protein bodies, large spherical, small spherical, and irregularly-shaped, were observed, in agreement with existing reports. Immunocytochemical studies showed the presence of glutelins in the irregularly-shaped protein bodies while the prolamines were found in both the large and small spherical protein bodies. Both the large and small spherical protein bodies, distinguishable by electron density and gold-labeling patterns, appear to be formed by direct deposition of the newly formed proteins into the lumen of the rough endoplasmic reticulum (ER). In contrast, glutelin protein bodies are formed via the Golgi apparatus. Small electron-lucent vesicles are often found at one side of the Golgi. Electron-dense vesicles, whose contents are labeled by glutelin antibody-gold particles, are commonly observed at the distal side of the Golgi apparatus and fuse to form the irregularly shaped protein bodies in endosperm cells. These observations indicate that the transport of rice glutelins from their site of synthesis, the ER, to the site of deposition, the protein bodies, is mediated by the Golgi apparatus.

R gene-controlled host specificity in the legume–rhizobia symbiosis

Leguminous plants can enter into root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. An intriguing but still poorly understood property of the symbiosis is its host specificity, which is controlled at multiple levels involving both rhizobial and host genes. It is widely believed that the host specificity is determined by specific recognition of bacterially derived Nod factors by the cognate host receptor(s). Here we describe the positional cloning of two soybean genes Rj2 and Rfg1 that restrict nodulation with specific strains of Bradyrhizobium japonicum and Sinorhizobium fredii, respectively. We show that Rj2 and Rfg1 are allelic genes encoding a member of the Toll-interleukin receptor/nucleotide-binding site/leucine-rich repeat (TIR-NBS-LRR) class of plant resistance (R) proteins. The involvement of host R genes in the control of genotype-specific infection and nodulation reveals a common recognition mechanism underlying symbiotic and pathogenic host–bacteria interactions and suggests the existence of their cognate avirulence genes derived from rhizobia. This study suggests that establishment of a root nodule symbiosis requires the evasion of plant immune responses triggered by rhizobial effectors.

Nod factors of Rhizobium are a key to the legume door

Symbiotic interactions between rhizobia and legumes are largely controlled by reciprocal signal exchange. Legume roots excrete flavonoids which induce rhizobial nodulation genes to synthesize and excrete lopo‐oligosaccharide Nod factors. In turn, Nod factors provoke deformation of the root hairs and nodule primordium formation. Normally, rhizobia enter roots through infection threads in markedly curled root hairs. If Nod factors are responsible for symbiosis‐specific root hair deformation, they could also be the signal for entry of rhizobia into legume roots. We tested this hypothesis by adding, at inoculation, NodNGR‐factors to signal‐production‐deficient mutants of the broad‐host‐range Rhizobium sp. NGR234 and Bradyrhizobium japorticum strain USDA110. Between 10 −7 M and 10−6 M NodNGR factors permitted these NodABC mutants to penetrate, nodulate and fix nitrogen on Vigna unguiculata and Glycine max, respectively. NodNGR factors also allowed Rhizobium fredii strain USDA257 to enter and fix nitrogen on Calopogonium caeruleum, a non‐host. Detailed cytological investigations of V. unguiculata showed that the NodABC mutant UGR AnodABC, in the presence of NodNGR factors, entered roots in the same way as the wild‐type bacterium. Since infection threads were also present in the resulting nodules, we conclude that Nod factors are the signals that permit rhizobia to penetrate legume roots via infection threads.

Molecular cloning and characterization of a sym plasmid locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257

Rhizobium fredii strain USDA257 produces nitrogen‐fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposonmutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain U5DA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolWand nolX are of the opposite polarity. The initiation codon for nolW lies 155bp upstream from that of nolB, and it is separated from nolXby 281 bp. The predicted NolT and NolW proteins have putative membrane‐spanning regions. The N‐terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site‐directed mutagenesis with mudll1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU anti nolX is induced as much as 30‐fold by flavonoid signal molecules, even though these genes lack nod‐box promoters. Histochemical staining of McCall roots inoculated with nolB–, nolU–, or nolX–lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU–ORF4–nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad‐host‐range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.

Extracellular proteins involved in soybean cultivar-specific nodulation are associated with pilus-like surface appendages and exported by a type III protein secretion system in Sinorhizobium fredii USDA257

Several gram-negative plant and animal pathogenic bacteria have evolved a type III secretion system (TTSS) to deliver effector proteins directly into the host cell cytosol. Sinorhizobium fredii USDA257, a symbiont of soybean and many other legumes, secretes proteins called Nops (nodulation outer proteins) into the extracellular environment upon flavonoid induction. Mutation analysis and the nucleotide sequence of a 31.2-kb symbiosis (sym) plasmid DNA region of USDA257 revealed the existence of a TTSS locus in this symbiotic bacterium. This locus includes rhc (rhizobia conserved) genes that encode components of a TTSS and proteins that are secreted into the environment (Nops). The genomic organization of the TTSS locus of USDA257 is remarkably similar to that of another broad-host range symbiont, Rhizobium sp. strain NGR234. Flavonoids that activate the transcription of the nod genes of USDA257 also stimulate the production of novel filamentous appendages known as pili. Electron microscope examination of isolated pili reveals needle-like filaments of 6 to 8 nm in diameter. The production of the pili is dependent on a functional nodD1 and the presence of a nod gene-inducing compound. Mutations in several of the TTSS genes negate the ability of USDA257 to elaborate pili. Western blot analysis using antibodies raised against purified NopX, Nop38, and Nop7 reveals that these proteins were associated with the pili. Mutations in rhcN, rhcJ, rhcC, and ttsI alter the ability of USDA257 to form nodules on Glycine max and Macroptilium atropurpureum.

A Functional myo-Inositol Dehydrogenase Gene Is Required for Efficient Nitrogen Fixation and Competitiveness of Sinorhizobium fredii USDA191 To Nodulate Soybean (Glycine max [L.] Merr.)

Inositol derivative compounds provide a nutrient source for soil bacteria that possess the ability to degrade such compounds. Rhizobium strains that are capable of utilizing certain inositol derivatives are better colonizers of their host plants. We have cloned and determined the nucleotide sequence of the myo-inositol dehydrogenase gene (idhA) of Sinorhizobium fredii USDA191, the first enzyme responsible for inositol catabolism. The deduced IdhA protein has a molecular mass of 34,648 Da and shows significant sequence similarity with protein sequences of Sinorhizobium meliloti IdhA and MocA; Bacillus subtilis IolG, YrbE, and YucG; and Streptomyces griseus StrI. S. fredii USDA191 idhA mutants revealed no detectable myo-inositol dehydrogenase activity and failed to grow on myo-inositol as a sole carbon source. Northern blot analysis and idhA-lacZ fusion expression studies indicate that idhA is inducible by myo-inositol. S. fredii USDA191 idhA mutant was drastically affected in its ability to reduce nitrogen and revealed deteriorating bacteroids inside the nodules. The number of bacteria recovered from such nodules was about threefold lower than the number of bacteria isolated from nodules initiated by S. fredii USDA191. In addition, the idhA mutant was also severely affected in its ability to compete with the wild-type strain in nodulating soybean. Under competitive conditions, nodules induced on soybean roots were predominantly occupied by the parent strain, even when the idhA mutant was applied at a 10-fold numerical advantage. Thus, we conclude that a functional idhA gene is required for efficient nitrogen fixation and for competitive nodulation of soybeans by S. fredii USDA191.

Assembly of the Cysteine Synthase Complex and the Regulatory Role of Protein-Protein Interactions

Macromolecular assemblies play critical roles in regulating cellular functions. The cysteine synthase complex (CSC), which is formed by association of serine O-acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), acts as a sensor and modulator of thiol metabolism by responding to changes in nutrient conditions. Here we examine the oligomerization and energetics of formation of the soybean CSC. Biophysical examination of the CSC by size exclusion chromatography and sedimentation ultracentrifugation indicates that this assembly (complex Mr ∼ 330,000) consists of a single SAT trimer (trimer Mr ∼ 110,000) and three OASS dimers (dimer Mr ∼ 70,000). Analysis of the SAT-OASS interaction by isothermal titration calorimetry reveals negative cooperativity with three distinct binding events during CSC formation with Kd values of 0.3, 7.5, and 78 nm. The three binding events are also observed using surface plasmon resonance with comparable affinities. The stability of the CSC derives from rapid association and extremely slow dissociation of OASS with SAT and requires the C terminus of SAT for the interaction. Steady-state kinetic analysis shows that CSC formation enhances SAT activity and releases SAT from substrate inhibition and feedback inhibition by cysteine, the final product of the biosynthesis pathway. Cysteine inhibits SAT and the CSC with Ki values of 2 and 70 μm, respectively. These results suggest a new model for the architecture of this regulatory complex and additional control mechanisms for biochemically controlling plant cysteine biosynthesis. Based on previous work and our results, we suggest that OASS acts as an enzyme chaperone of SAT in the CSC.

Biochemistry and Molecular Biology of Soybean Seed Storage Proteins

ABSTRACT Soybean (Glycine max[L.] Merr.) is one of the principle field crops grown in the United States. From a crop with no significant economic value at the turn of this century, soybeans have made remarkable strides in U.S. agriculture, claiming the stature of the number two U.S. cash crop. Soybean is an important source of edible vegetable oil and protein throughout the world and is used in a multitude of food and industrial applications. Even though soybeans are a rich source of protein for livestock and humans, the nutritional quality of soybean proteins is not optimal. Some of the problems associated with soybean proteins include (1) presence of anti-nutritional factors such as trypsin inhibitor, (2) undesirable beany flavor, (3) elicitation of allergic reactions in susceptible individuals, (4) poor digestibility of soybean proteins, and (5) deficiency in sulfur-containing amino acids. As a consequence, concerted efforts are underway to improve the overall nutritive value of soybean proteins by both classical plant breeding and molecular biological approaches. This review article summarizes the current knowledge on the biochemistry and molecular biology of soybean seed storage proteins. Recent advances in the genetic improvement of amino acid composition of seed storage proteins are highlighted. The review also includes some recent achievements in modifying soybean seed composition.

Nodulation of Sesbania species by Rhizobium (Agrobacterium) strain IRBG74 and other rhizobia.

Summary Concatenated sequence analysis with 16S rRNA, rpoB and fusA genes identified a bacterial strain (IRBG74) isolated from root nodules of the aquatic legume Sesbania cannabina as a close relative of the plant pathogen Rhizobium radiobacter (syn. Agrobacterium tumefaciens). However, DNA:DNA hybridization with R. radiobacter, R. rubi, R. vitis and R. huautlense gave only 44%, 5%, 8% and 8% similarity respectively, suggesting that IRBG74 is potentially a new species. Additionally, it contained no vir genes and lacked tumour‐forming ability, but harboured a sym‐plasmid containing nifH and nodA genes similar to those in other Sesbania symbionts. Indeed, IRBG74 effectively nodulated S. cannabina and seven other Sesbania spp. that nodulate with Ensifer (Sinorhizobium)/Rhizobium strains with similar nodA genes to IRBG74, but not species that nodulate with Azorhizobium or Mesorhizobium. Light and electron microscopy revealed that IRBG74 infected Sesbania spp. via lateral root junctions under flooded conditions, but via root hairs under non‐flooded conditions. Thus, IRBG74 is the first confirmed legume‐nodulating symbiont from the Rhizobium (Agrobacterium) clade. Cross‐inoculation studies with various Sesbania symbionts showed that S. cannabina could form fully effective symbioses with strains in the genera Rhizobium and Ensifer, only ineffective ones with Azorhizobium strains, and either partially effective (Mesorhizobium huakii) or ineffective (Mesorhizobium plurifarium) symbioses with Mesorhizobium. These data are discussed in terms of the molecular phylogeny of Sesbania and its symbionts.

Formation of wheat protein bodies: involvement of the Golgi apparatus in gliadin transport

Developing wheat (Triticum aestivum L.) endosperm was examined using ultrathin sections prepared from tissues harvested at 5, 9, 16 and 25 d after flowering. Protein bodies were evident by 9 d and displayed a variety of membranous structures and inclusions. The Golgi apparatus was a prominent organelle at all stages, and by 9 d was associated with small electron-dense inclusions. By immunocytochemical techniques, gliadin (wheat prolamine) was localized within these vesicles and in homogeneous regions of protein bodies, but not in the lumen of the rough endoplasmic reticulum. The protein bodies appear to enlarge by fusion of smaller protein bodies resulting in larger, irregular-shaped organelles. The affinity of the Golgi-derived vesicles for gliadin-specific probes during the period of maximal storage-protein synthesis and deposition indicates that this organelle includes the bulk, if not all, of the gliadin produced. The involvement of the Golgi apparatus in the packaging of gliadins into protein bodies indicates a pathway which differs from the mode of prolamine deposition in other cereals such as maize, rice and sorghum, and resembles the mechanism employed for the storage of rice glutelin and legume globulins.