Changes of Thylakoid Membrane Stacks and Chl a/b Ratio of Chloroplast from Sacred Lotus (Nelumbo nucifera) Seeds During Their Germination Under Light

Changes of chloroplast thylakoid membrane stacks and Chl a/b ratio in the plumule of sacred lotus (Nelumbo nucifera Gaertn) seeds during their germination under light were as follows: Before germination there were giant grana and very low Chi a/b ratio (0.9) in the chloroplasts. Two days after germination, the thylakoid membranes of the giant grana gradually loosened and even destacked (disintegrated), the Chl a/b ratio was 1.06. Four clays after germination, the newly formed grana thylakoid membranes were 3–5 times shorter than those of the supergrana thylakoid membranes before germination and less grana stacks were seen; the Chl a/b ratio was 1.42. Six days after germination, the stacked thylakoi membranes became more orderly arranged. In addition the grana increased in number, the stroma thylakoid membranes were scarce, the Chl a/b ratio was 2.16. Eiglt days after germination, the thylakoid membranes in each granum decreased, but the total number of grana increased only slightly. In the meantime, some large starch grains and more stroma thylakoid membranes appeared; the Chl a/b ratio was 2.77. Ten days after germination normal thylakoid membrane structure was formed both in grana and stroma lamellae. They were arranged orderly as in the chloroplasts of other higher plants; the Chl a/b ratio was 2.80. The following conclusions could be drawn from the above mentioned results: 1) There was a negative correlation between the degree of stacking of the grana thylakoid membranes and the Chl a/b ratio. This statement further proved that the membranes stacking might mainly be induced by LHCII. 2) Development of the grana thylakoid membranes within chloroplasts from sacred lotus plumule followed that of the stroma thylakoid membranes, and the tendency of changes of their Chl 2/b ratio being from the lowest to the highest and then to normal were quite different from those of other higher plants. The chloroplasts iri the latter plants contain long parallel stacks of nonappressed primary thylakoids at second step, and the changes of their ratio of Chl a/b tend to be from the highest to the lowest and then to normal. There are indications that sacred lotus plumule might employ a distinctive developing pathway. This provides an important basis for Nelumbo to possess an unique position in phylogeny of Angiospermae.     

Chromosomes of Six Fabaceous Species from Baoxing County,Sichuan Province

Meiosis and/or mitosis of six species of Fabaceae (Leguminosae) from Baoxing County, Sichuan, China, were investigated. The voucher specimens are conserved in PE. Eight pairs (n=8) and 10 chiasmata in meiosis of pollen mother cells have been observed in Medicago lupulina L. (Pl. 1, A-C). Meiotic observation on pollen mother cells in Lotus tenuis W. et K. shows 6 bivalents (n=6) in MI and 9 chiasmata in diakinesis (Pl. 1, D-E). In this species 12 somatic chromosomes (2n=12) in anther wall cells have also been observed. The chromosomal formula may be expressed as 2n=12=8m+2sm+2sm^SAT (Pl. 1, F-G). In pollen mother cells of Vicia tetrasperma (L.) Schreb., 7 bivalents in MI and 7 chromosomes in A II have been observed (Pl. 2, A-B). From A II (Pl. 2, B, the inset on the right) the chromosomal formula, n=7= 2m+2sm+lst^SAT+2t, may be constructed. Only three chromosomes in this karyotype may be found to have counterparts in the one reported by Srivastava (1963), which shows striking differences between these two karyotypes. Meiotic MI shows 7 pairs (n=7) in Vicia hirsuta (L.) S. F. Gray. Vicia sativa L. is very variable in its chromosomes. Our observation shows 6 pairs (n=6) in MI and in diakinesis in pollen mother cells. In Vicia villosa Roth, all the previous chromosome reports are 2n=14 or n=7, but the result of our work shows that somatic chromosomes are 2n=12 in anther wall cells (Pl. 3, D, E). The karyotype in our material (Pl. 3, E) is that the longest pair of chromosomes are metacentric, the pairs 2-4 are terminal, 5 are metacentric and last pair are submetacentric, differing vastly from the idiogram (Pl. 3, F) presented by Yamamoto (1973). Therefore both the chromosome number and structure in our material are greatly different from those in all the previous reports. The evolutionary trends of chromosomes in the genus Vicia is discussed in the work. Srivastava (1963) holds that the primitive basic number of chromosome in the genus is 6 and thus both 5 and 7 are derived. The present author would propose another possibility that 7 is the original basic number and the other numbers are derived ones. First, as shown in Table 1, x=7 occurs in 47 per cent of species in the genus, but 6 only in 28 per cent. Secondly, x=7 is predominant in the perennial and primitive section Cracca. Thirdly, in genera related to the genus under consideration, such as Lens, Pisum and Lathyrus, x=7 is also the predominant basic number. Fourthly, according to Raven (1975) 7 is the primitive basic number in the angiosperms and x= 7, 8 and 9 are the predominant in the angiosperms.     

N‐glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research

Studies of protein N‐glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N‐glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N‐glycan patterns as documented using mass spectrometry and glycan‐recognising antibodies, indicating successful identification of null mutations in the target glyco‐genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3‐fucosyltransferase (Lj3fuct) mutant completely lacked α1,3‐core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N‐glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N‐acetylglucosaminyltransferase I, and α1,3‐fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N‐glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N‐glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian‐like N‐glycosylation features.     

LjMOT1, a high‐affinity molybdate transporter from Lotus japonicus,is essential for molybdate uptake,but not for the delivery to nodules

Molybdenum (Mo) is an essential nutrient for plants, and is required for nitrogenase activity of legumes. However, the pathways of Mo uptake from soils and then delivery to the nodules have not been characterized in legumes. In this study, we characterized a high‐affinity Mo transporter (LjMOT1) from Lotus japonicus. Mo concentrations in an ethyl methanesulfonate–mutagenized line (ljmot1) decreased by 70–95% compared with wild‐type (WT). By comparing the DNA sequences of four AtMOT1 homologs between mutant and WT lines, one point mutation was found in LjMOT1, which altered Trp²⁹² to a stop codon; no mutation was found in the other homologous genes. The phenotype of Mo concentrations in F₂ progeny from ljmot1 and WT crosses were associated with genotypes of LjMOT1. Introduction of endogenous LjMOT1 to ljmot1 restored Mo accumulation to approximately 60–70% of the WT. Yeast expressing LjMOT1 exhibited high Mo uptake activity, and the K_m was 182 nm . LjMOT1 was expressed mainly in roots, and its expression was not affected by Mo supply or rhizobium inoculation. Although Mo accumulation in the nodules of ljmot1 was significantly lower than that of WT, it was still high enough for normal nodulation and nitrogenase activity, even for cotyledons‐removed ljmot1 plants grown under low Mo conditions, in this case the plant growth was significantly inhibited by Mo deficiency. Our results suggest that LjMOT1 is an essential Mo transporter in L. japonicus for Mo uptake from the soil and growth, but is not for Mo delivery to the nodules.     

Alternative temperate forages containing secondary compounds for improving sustainable productivity in grazing ruminants

The use of alternative temperate forages to improve the sustainable productivity of grazing ruminants, relative to grass-based pastures, is reviewed. Particular emphasis is placed upon forages containing secondary compounds for sustainable control of internal parasites, for increasing reproductive rate in sheep, reducing bloat risk in cattle and for reducing methane production as a means of lowering greenhouse gas emissions.

Of the forages reviewed, the herb chicory (Chicorium intybus) and the condensed tannin-containing legumes Lotus corniculatus L. and sulla (Hedysarum coronarium) offered the most advantages. Chicory and sulla promoted faster growth rates in young sheep and deer in the presence of internal parasites, and showed reduced methane production in other studies. L. corniculatus was not as effective as chicory and sulla in promoting growth of lambs in the presence of internal parasites. Grazing on L. corniculatus was associated with increases in reproductive rate in sheep, increases in milk production in both ewes and dairy cows and reduced methane production, effects that were mainly due to its content of condensed tannins (CT). Grazing ewes on L. corniculatus during mating and very early pregnancy may also reduce lamb mortality. However, there are no data on the effect of mating ewes, which are grazing chicory on their reproductive performance, an important omission. Risk of rumen frothy bloat in cattle grazing legumes is reduced when the forage contains 5 g CT/kg dry matter (DM) or greater. Gene transfer techniques aimed at achieving this for lucerne (Medicago sativa) have made progress, but CT concentration needs to be further increased from calculated values of 0.75–1.25 g CT/kg DM in the transformed plants. Bloat control may be achievable in genetically transformed legumes before increased amino acid absorption, as the concentration of CT required for bloat control is lower (5 versus 30–40 g/kg DM) than that required to cause increased amino acid absorption and is not affected by differences in CT structure.

Key plant characteristics for improved sustainable productivity are a high ratio of readily fermentable: structural carbohydrate and the presence of CT and certain other secondary compounds.

Taking into account both nutritional and agronomic considerations, chicory is considered one of the best emerging plants for grazing livestock, with L. corniculatus being more suitable for areas with dry summers and warm winters. Some of the agronomic limitations of L. corniculatus and sulla could be reduced by mechanical harvesting and their inclusion as a component in total mixed rations (TMR), instead of grazing.     

Cumulative effects of fire on a tussock pampa grassland

Abstract. In order to test for cumulative effects of fire on Paspalum quadrifarium‐dominated grasslands (‘pajonal’), we analysed the impact of single and repeated fires on the community structure and post‐fire recovery of canopy after a final, simultaneous fire event. Nine plots were defined within a homogeneous pajonal stand, and treatments of low (LF), medium (MF) and high frequency (HF) of fire were defined by the application of one, two or four cold‐season burns, respectively, along a 6‐yr period. Both burned and unburned plots were exposed to grazing by cattle during the summer following the first and the third years of that period. High cattle preference for burned sites conditioned fire temperature and vegetation responses to the following burning events. Cumulative effects between successive burning events were observed for the cover of basal area of the dominant and other sprouting species, the cover and thickness of the litter layer, the seed bank size of the principal recruiter species, and the floristic composition. While light interception by the canopy was positively related to fire frequency during the early growth season, further growth of P. quadrifarium determined a greater light interception in LF than in MF and HF. These patterns of light interception were associated with a faster occupation of the inter‐tussock areas by opportunistic species in plots subjected to frequent fires (HF and MF) than in plots with low fire frequency (LF), and a more lasting regrowth of P. quadrifarium in the LF plot than in the HF ones. High fire frequencies reduced the dominance of P. quadrifarium. Percent of species classified as subordinated graminoids or forbs did not vary among treatments. However, the abundance of different forb species was differentially favoured by contrasting frequencies of fire, describing some coarse relationships between their specific responses and their dispersal strategies.     

Development and characterization of simple sequence repeat (SSR) markers in the water lotus (Nelumbo nucifera)

Simple sequence repeat (SSR) markers were developed in the water lotus (Nelumbo nucifera Gaertn.) from an SSR-enriched genomic library. Of the SSR markers tested, 11 primer pairs produced clearly distinguishable DNA banding patterns. Forty-three alleles were detected with the 11 markers. The allele number per locus ranged from 2 to 5 with an average of 3.9. Polymorphism values ranged from 0.11 to 0.66 with an average of 0.51. These primers were also applicable to another Nelumbo species, Nelumbo lutea (Willd.) Pers. (American lotus) and hybrids between N. nucifera and N. lutea. These results indicate that the SSR markers developed in this study are informative and will be useful for genetic analysis in Nelumbo species.     

Nod Factor/Nitrate-Induced CLE Genes that Drive HAR1-Mediated Systemic Regulation of Nodulation

Host legumes control root nodule numbers by sensing externaland internal cues. A major external cue is soil nitrate, whereasa feedback regulatory system in which earlier formed nodulessuppress further nodulation through shoot–root communicationis an important internal cue. The latter is known as autoregulationof nodulation (AUT), and is believed to consist of two long-distancesignals: a root-derived signal that is generated in infectedroots and transmitted to the shoot; and a shoot-derived signalthat systemically inhibits nodulation. In Lotus japonicus, theleucine-rich repeat receptor-like kinase, HYPERNODULATION ABERRANTROOT FORMATION 1 (HAR1), mediates AUT and nitrate inhibitionof nodulation, and is hypothesized to recognize the root-derivedsignal. Here we identify L. japonicus CLE-Root Signal 1 (LjCLE-RS1)and LjCLE-RS2 as strong candidates for the root-derived signal.A hairy root transformation study shows that overexpressingLjCLE-RS1 and -RS2 inhibits nodulation systemically and, furthermore,that the systemic suppression depends on HAR1. Moreover, LjCLE-RS2expression is strongly up-regulated in roots by nitrate addition.Based on these findings, we propose a simple model for AUT andnitrate inhibition of nodulation mediated by LjCLE-RS1, -RS2peptides and the HAR1 receptor-like kinase.     

百脉根基因工程研究进展

牧草基因工程是近年来国内外研究的热点之一.针对农杆菌介导百脉根遗传转化原理、影响农杆菌介导百脉根遗传转化的重要因素、转基因技术在百脉根的生物固氮、抗逆性和品质改良以及生产可食性疫苗等方面的研究进行了全面综述,并就百脉根基因工程研究今后的主要发展方向进行了展望.     

Comparative ionomics and metabolomics in extremophile and glycophytic Lotus species under salt stress challenge the metabolic pre-adaptation hypothesis

The legume genus Lotus includes glycophytic forage crops and other species adapted to extreme environments, such as saline soils. Understanding salt tolerance mechanisms will contribute to the discovery of new traits which may enhance the breeding efforts towards improved performance of legumes in marginal agricultural environments. Here, we used a combination of ionomic and gas chromatography‐mass spectrometry (GC‐MS)‐based metabolite profilings of complete shoots (pooling leaves, petioles and stems) to compare the extremophile Lotus creticus, adapted to highly saline coastal regions, and two cultivated glycophytic grassland forage species, Lotus corniculatus and Lotus tenuis. L. creticus exhibited better survival after exposure to long‐term lethal salinity and was more efficient at excluding Cl^‐ from the shoots than the glycophytes. In contrast, Na⁺ levels were higher in the extremophile under both control and salt stress, a trait often observed in halophytes. Ionomics demonstrated a differential rearrangement of shoot nutrient levels in the extremophile upon salt exposure. Metabolite profiling showed that responses to NaCl in L. creticus shoots were globally similar to those of the glycophytes, providing little evidence for metabolic pre‐adaptation to salinity. This study is the first comparing salt acclimation responses between extremophile and non‐extremophile legumes, and challenges the generalization of the metabolic salt pre‐adaptation hypothesis.