Physiological,biochemical, and molecular differences in chloroplast synthesis between leaf and corolla of cabbage (<Emphasis Type="Italic">Brassica rapa</Emphasis> L. var. chinensis) and rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

Cadmium (Cd) pollution is one of the major concerns in the development of sustainable agriculture, particularly for rice production. Silicon (Si) was recently recognized for its ability to mitigate a variety of abiotic stresses including that caused by Cd on rice. However, mechanism of the complex process is still not fully understood. Under Cd-stress, the low Si-influx 1-RNAi transgenic Lemont rice (Lsi1-RNAi line) exhibited an increased Cd-uptake over its counterparts, the wild type and the Lsi1-overexpression transgenic rice (Lsi1-OE line). In contrast, the Lsi1 expression-enhanced Lsi1-OE line showed the greatest Si-uptake among the three lines, with the highest activities on anti-oxidants (such as, superoxide dismutase) and the lowest content of malondialdehyde. Lsi1 also displayed a negative regulation on Low Cd gene and the natural resistance-associated macrophage proteins, Nramp5, indicating its capacity to alleviate Cd-stress on rice. The results obtained by this study suggested that the mitigation of Cd-toxicity on rice by Si might involve functions, such as the inhibition on Cd-uptake and transport and the enhancement on anti-oxidative enzyme activities, as well as the Lsi1-related expression on regulation of Si-uptake in rice. A new avenue might become available for overcoming the rampant pollution that threatens the rice production in China.     

<Emphasis Type="Italic">Lsi1</Emphasis>-regulated Cd uptake and phytohormones accumulation in rice seedlings in presence of Si

Silicon (Si) is known for its role in regulating the response of plants to imposed abiotic stresses. Since the stresses generally hinder production of a crop, such as rice, the exploration of the biochemistry and plant physiology relating to the function is of interest. Indeed, recently, there were reports on the function of Lsi1 in regulating the tolerance of rice to cadmium (Cd) stress. This study compared the kinetics of the Cd uptakes in Lemont wild type rice and its transgenic lines exposed to Cd with or without exogenous Si supply. At the same time, changes on the endogenous phytohormones and growth of the rice seedlings were monitored. Genetically, Lsi1 overexpression was found to downregulate K_m and V_max of Cd uptake kinetics in the plants under Cd stress, especially in the presence of Si. On the other hand, Lsi1 RNAi upregulated K_m and V_max regardless whether Si was present or not. It implied that Lsi1 could be capable of regulating Si as well as Cd transports. Under Cd stress, addition of Si reduced the Cd uptake of the rice lines in the order of Lsi1-overexpression line?>?Lemont?>?Lsi1-RNAi line. In addition, it also affected the chlorophyll biosynthesis and dry mass accumulation of the rice plants under Cd stress. Analyses on phytohormones including IAA, GA₃, JA, SA and ABA, as well as physiological functions, of the seedlings further verified the active involvement of Lsi1 in the complex defense system of the plants against Cd stress.     

Characterisation of a novel quantitative trait locus, <Emphasis Type="Italic">GN4</Emphasis>-<Emphasis Type="Italic">1</Emphasis>, for grain number and yield in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Key message

A novel QTL for grain number, GN4-1, was identified and fine-mapped to an ~ 190-kb region on the long arm of rice chromosome 4.

Abstract

Rice grain yield is primarily determined by three components: number of panicles per plant, grain number per panicle and grain weight. Among these traits, grain number per panicle is the major contributor to grain yield formation and is a crucial trait for yield improvement. In this study, we identified a major quantitative trait locus (QTL) responsible for rice grain number on chromosome 4, designated GN4-1 (a QTL for Grain Number on chromosome 4), using advanced segregating populations derived from the crosses between an elite indica cultivar ‘Zhonghui 8006’ (ZH8006) and a japonica rice ‘Wuyunjing 8’ (WYJ8). GN4-1 was delimited to an ~ 190-kb region on chromosome 4. The genetic effect of GN4-1 was estimated using a pair of near-isogenic lines. The GN4-1 gene from WYJ8 promoted accumulation of cytokinins in the inflorescence and increased grain number per panicle by ~ 17%. More importantly, introduction of the WYJ8 GN4-1 gene into ZH8006 increased grain yield by ~ 14.3 and ~ 11.5% in the experimental plots in 2014 and 2015, respectively. In addition, GN4-1 promoted thickening of the culm and may enhance resistance to lodging. These results demonstrate that GN4-1 is a potentially valuable gene for improvement of yield and lodging resistance in rice breeding.

     

Tolerance to soil water stress by <Emphasis Type="Italic">Oryza sativa</Emphasis> cv. IR20 was improved by expression of <Emphasis Type="Italic">Wsi18</Emphasis> gene locus from <Emphasis Type="Italic">Oryza nivara</Emphasis>

Wild rice genotypes are rich in genetic diversity. This has potential to improve agronomic rice by allele mining for superior traits. Late embryogenesis abundant (LEA) proteins are often associated with desiccation tolerance and stress signalling. In the present study, a group 3 LEA gene, Wsi18 from the wild rice Oryza nivara was expressed under its own inducible promoter element in stress susceptible cultivated indica rice (cv. IR20). The resulting transgenic plants cultivated in a greenhouse showed enhanced tolerance to soil water deficit. Transgenic plants had higher grain yield, plant survival rate, and shoot relative water content compared to wild type (WT) IR20. Cell membrane stability index, proline and soluble sugar content were also greater in transgenic than WT plants under water stress. These results demonstrate the potential for improving SWS tolerance in agronomically important rice cultivar by incorporating Wsi18 gene from a wild rice O. nivara.     

Genetic dissection of large grain shape in rice cultivar ‘Nanyangzhan’ and validation of a grain thickness QTL (<Emphasis Type="Italic">qGT3.1</Emphasis>) and a grain length QTL (<Emphasis Type="Italic">qGL3.4</Emphasis>)

Grain shape is an important agronomic trait in rice, which influences the yield and quality. In order to dissect the genetic basis of the large grain shape in ‘Nanyangzhan’, a recombinant inbred line (RIL) population derived from Nanyangzhan (NYZ) and Zhenshan 97B (ZS97) was used to map quantitative trait loci (QTLs) for grain length (GL), width (GW), thickness (GT), length-to-width ratio (LWR) and kilo-grain weight (KGW). A total of 53 QTLs were detected and distributed on 11 chromosomes in 2 years. Among those, QTLs for GW and GL showed a concentrated distribution on chromosome 2 and chromosome 3, respectively. NYZ, the parent with large grain shape, carried 44 alleles showing positive effects on the studied traits. In addition, the near-isogenic lines (NILs) of two novel QTLs, qGT3.1 and qGL3.4, were constructed with the background of ZS97. Results showed that NIL-qGT3.1 ^NYZ , the NIL carrying homozygous qGT3.1 regions from NYZ, showed an increased value of 0.12 mm in grain thickness on average as compared to NIL-qGT3.1 ^ZS . Similarly, NIL-qGL3.4 ^NYZ increased the length of each grain by 0.47 mm on average as compared to NIL-qGL3.4 ^ZS . Taken together, these results would be of great use in breeding rice cultivars with desirable grain shape.     

Transgenic rice expressing the <Emphasis Type="Italic">cry</Emphasis>2AX1 gene confers resistance to multiple lepidopteran pests

A chimeric Bacillus thuringiensis toxin (Bt) gene, cry2AX1was cloned in a bi-selectable marker free binary vector construct. The cry2AX1 gene, driven by the Chrysanthemum rbcS1 promoter, was introduced into JK1044R, the restorer line (Oryza sativa L. ssp. Indica) of a notified commercially grown rice hybrid in India, by Agrobacterium-mediated transformation. Its effect against two major lepidopteran insect pests viz., yellow stem borer (YSB) Scirpophaga incertulas, rice leaf folder (RLF) Cnaphalocrocis medinalis and one minor insect pest, oriental army worm (OAW) Mythimna separata was demonstrated through bioassays of transgenic rice plants under laboratory and greenhouse conditions. The rbcS1 promoter with chloroplast signal peptide was used to avoid Cry2AX1 protein expression in rice seed endosperm tissue. A total of 37 independent transformants were generated, of which after preliminary molecular characterization and YSB bioassay screening, five events were selected for their protein expression and bioefficacy against all three rice insect. One elite transgenic rice line, BtE15, was identified with Cry2AX1 expression ranging from 0.68 to 1.34 µg g^?1 leaf fresh weight and with 80–92 % levels of resistance against rice pests at the vegetative and reproductive stages. Increase in Cry2AX1 protein concentration was also observed with crop maturity. The Cry2AX1protein concentration in the de-husked seeds was negligible (as low as 2.7–3.6 ng g^?1). These results indicate the potential application of cry2AX1 gene in rice for protection against YSB, RLF and OAW.     

Constitutive expression of a plant ferredoxin-like protein (pflp) enhances capacity of photosynthetic carbon assimilation in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

The plant ferredoxin-like protein (PFLP) gene, cloned from sweet peppers predicted as an electron carrier in photosynthesis, shows high homology to the Fd-I sequence of Arabidopsis thaliana, Lycopersicon esculentum, Oryza sativa and Spinacia oleracea. Most of pflp related studies focused on anti-pathogenic effects, while less understanding for the effects in photosynthesis with physiological aspects, such as photosynthesis rate, and levels of carbohydrate metabolites. This project focuses on the effects of pflp overexpression on photosynthesis by physiological evaluations of carbon assimilation with significant higher levels of carbohydrates with higher photosynthesis efficiency. In this report, two independent transgenic lines of rice plants (designated as pflp-1 and pflp-2) were generated from non-transgenic TNG67 rice plant (WT). Both transgenic pflp rice plants exhibited enhanced photosynthesis efficiency, and gas exchange rates of photosynthesis were 1.3- and 1.2-fold higher for pflp-1 and pflp-2 than WT respectively. Significantly higher electron transport rates of pflp rice plants were observed. Moreover, photosynthetic products, such as fructose, glucose, sucrose and starch contents of pflp transgenic lines were increased accordingly. Molecular evidences of carbohydrate metabolism related genes activities (osHXK5, osHXK6, osAGPL3, osAGPS2α, osSPS, ospFBPase, oscFBPase, and osSBPase) in transgenic lines were higher than those of WT. For performance of crop production, 1000-grain weight for pflp-1 and pflp-2 rice plants were 52.9 and 41.1 g that were both significantly higher than 31.6 g for WT, and panicles weights were 1.4- and 1.2-fold higher than WT. Panicle number, tiller number per plants for pflp rice plants were all significantly higher compared with those of WT where there was no significant difference observed between two pflp rice plants. Taken altogether; this study demonstrated that constitutive pflp expression can improve rice production by enhancing the capacity of photosynthetic carbon assimilation.     

Genetic mapping and salt tolerance of a novel <Emphasis Type="Italic">D1</Emphasis>-allelic mutant of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plant height and grain shape are important traits that may affect yield in rice, and they therefore have enormous importance in breeding. A dwarf small-grain mutant (S525) was identified among progeny of the Indica rice restorer line ‘Xida 1B’ (wild type) raised from seeds treated with ethyl methanesulfonate. The dwarf and small-grain phenotypes were stably inherited after multi-generation selfing. Field-grown mutant plants showed the phenotypes of dwarfism, broad leaves, and small round grains. Genetic mapping and sequencing confirmed that S525 was a novel d1-allelic mutant. A single-base transition (G to A) in the functional dwarfism gene D1 at the conjunction site of the 11th intron caused excision or duplication of the 11th exon in the mRNA and resulted in translation of a defective G_α protein. The S525 showed enhanced salt tolerance compared with the wild type (WT), and the expression of genes associated with salt tolerance quantitatively increased in response to treatment with 200 mM NaCl. The S525 may be useful for future investigation of G_α functions and in the breeding of new dwarf rice cultivars.     

Over-expression of tobacco <Emphasis Type="Italic">UBC1</Emphasis> encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca²⁺/H⁺ exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.     

Expression of sorghum gene <Emphasis Type="Italic">SbSGL</Emphasis> enhances grain length and weight in rice

Grain weight is a major determining factor of rice (Oryza sativa L.) yield and the comprehensive embodiment of grain length, width, and thickness. Here, we describe the molecular and functional characterization of SbSGL (Sorghum bicolor L. stress tolerance and grain length), a sorghum gene that encodes a putative member of the DUF1645 protein family of unknown function. Expression of SbSGL in rice promoted cell division and grain filling, which affected an array of traits of rice, including grain length, grain weight, and seed setting rate. Expression of SbSGL also affected the expression of genes related to the plant cell cycle and grain size.     

The causal deletions in the second exon of <Emphasis Type="Italic">An-3</Emphasis> closely associated with awn development and rice yield

Awn is one of important traits during rice domestication. To understand the development of rice awn and the roles it played in rice domestication, we preliminary mapped a major QTL An-3 for awn development using chromosome segment substitution line CSSL138 developed by introgressed genomic fragments of long-awned Guangxi common wild rice (GXCWR, Oryza rufipogon Griff.) into genetic background of short-awned indica cultivar 93–11. An-3 was then fine mapped to a 7-kb region of chromosome 8. An epidermal patterning factor-like protein gene was identified as the single candidate gene corresponding to this QTL. An-3 was showed to be an allele of RAE2 and GAD1, and negatively regulated 1000-grains weight, grain length, and length–width ratio. Comparing with the coding sequences of An-3 from CSSL138, a 2- and 4-bp frame-shift deletions in the second exon were identified in 93–11 and Nipponbare, respectively. Taken together, our results provide valuable natural variation in the alleles of An-3 between common wild rice and cultivated rice, which will be helpful in clarifying the mechanism of awn development and promoting the application of an-3 in genetic improvement of rice yield traits.     

Darwin’s legacy in <Emphasis Type="Italic">Platanthera</Emphasis>: are there more than two species in the <Emphasis Type="Italic">Platanthera bifolia/chlorantha</Emphasis> group?

In Central Europe, the genus Platanthera traditionally comprised two species, P. chlorantha Cust. ex Rchb. (Pc) and P. bifolia (L.) Rich. (Pb). They are morphologically characterized by a wide and narrow separation of anthers, respectively. However, a third form with intermediate anther distance has repeatedly been hypothesized but only hesitantly accepted. In addition, intermediate morphology has been also used as the main character of P. × hybrida. However, the status of some purported hybrid populations is challenged by the local lack of parental species, their successful reproduction and non-intermediate traits. Despite this unclear situation, detailed genetic and morphological analyses are lacking. Here, we studied morphology and molecular markers within the P. chlorantha/bifolia group in Central Europe. Three morphological groups emerged representing Pc, Pb and a third form, here informally referred to as non-hybrid intermediates (Pn). The latter is characterized, among other trait differences, by intermediate distance between anthers [(0.7)–1–2.2 mm] and long spurs (28–40 mm). Three gene pools were identified, which largely corresponded to the three morphological groups. The Pn gene pool had several high-frequency private alleles substantiating its genetic independence. Some of the Pn populations were previously interpreted as P. × hybrida suggesting that Pn was overlooked hitherto and mistaken to represent hybrids. The non-perfect fit between morphological and genetic groups highlights the potential for fast morphological evolution. Overall, the finding of three distinct lineages within the bifolia/chlorantha group necessitates a thorough reanalysis of reported taxa and a reevaluation of our understanding of their distribution, ecology and evolution.     

<Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of commercially elite rice restorer line using <Emphasis Type="Italic">npt</Emphasis>II gene as a plant selection marker

Transformation of commercially important indica cultivars remains challenging for the scientific community even though Agrobacterium-mediated transformation protocols for a few indica rice lines have been well established. We report successful transformation of a commercially important restorer line JK1044R of indica rice hybrid JKRH 401. While following existing protocol, we optimized several parameters for callusing, regeneration and genetic transformation of JK1044R. Calli generated from the rice scutellum tissue were used for transformation by Agrobacterium harboring pCAMBIA2201. A novel two tire selection scheme comprising of Geneticin (G418) and Paramomycin were deployed for selection of transgenic calli as well as regenerated plantlets that expressed neomycin phosphotransferase-II gene encoded by the vector. One specific combination of G418 (30 mg l^?1) and Paramomycin (70 mg l^?1) was very effective for calli selection. Transformed and selected calli were detected by monitoring the expression of the reporter gene uidA (GUS). Regenerated plantlets were confirmed through PCR analysis of nptII and gus genes specific primers as well as dot blot using gus gene specific as probe.