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.     

The oxalyl‐CoA synthetase‐regulated oxalate and its distinct effects on resistance to bacterial blight and aluminium toxicity in rice

• Oxalic acid is widely distributed in biological systems and known to play functional roles in plants. The gene AAE3 was recently identified to encode an oxalyl‐CoA synthetase (OCS) in Arabidopsis that catalyses the conversion of oxalate and CoA into oxalyl‐CoA. It will be particularly important to characterise the homologous gene in rice since rice is not only a monocotyledonous model plant, but also a staple food crop.
• Various enzymatic and biological methods have been used to characterise the homologous gene.
• We first defined that AAE3 in the rice genome (OsAAE3) also encodes an OCS enzyme. Its K_m for oxalate is 1.73 ± 0.12 mm , and V_m is 6824.9 ± 410.29 U·min^?1·mg protein^?1. Chemical modification and site‐directed mutagenesis analyses identified thiols as the active site residues for rice OCS catalysis, suggesting that the enzyme might be regulated by redox state. Subcellular localisation assay showed that the enzyme is located in the cytosol and predominantly distributed in leaf epidermal cells. As expected, oxalate levels increased when OCS was suppressed in RNAi transgenic plants. More interestingly, OCS‐suppressed plants were more susceptible to bacterial blight but more resistant to Al toxicity.
• The results demonstrate that the OsAAE3‐encoded protein also acts as an OCS in rice, and may play different roles in coping with stresses. These molecular, enzymatic and functional data provide first‐hand information to further clarify the function and mechanism of OCS in rice plants.

     

Allantoin accumulation through overexpression of ureide permease1 improves rice growth under limited nitrogen conditions

In legumes, nitrogen (N) can be stored as ureide allantoin and transported by ureide permease (UPS) from nodules to leaves where it is catabolized to release ammonium and assimilation to amino acids. In non‐leguminous plants especially rice, information on its roles in N metabolism is scarce. Here, we show that OsUPS1 is localized in plasma membranes and are highly expressed in vascular tissues of rice. We further evaluated an activation tagging rice overexpressing OsUPS1 (OsUPS1^OX) under several N regimes. Under normal field conditions, panicles from OsUPS1^OX plants (14 days after flowering (DAF)) showed significant allantoin accumulation. Under hydroponic system at the vegetative stage, plants were exposed to N‐starvation and measured the ammonium in roots after resupplying with ammonium sulphate. OsUPS1^OX plants displayed higher ammonium uptake in roots compared to wild type (WT). When grown under low‐N soil supplemented with different N‐concentrations, OsUPS1^OX exhibited better growth at 50% N showing higher chlorophyll, tiller number and at least 20% increase in shoot and root biomass relative to WT. To further confirm the effects of regulating the expression of OsUPS1, we evaluated whole‐body‐overexpressing plants driven by the GOS2 promoter (OsUPS1^GOS2) as well as silencing plants (OsUPS1^RNAi). We found significant accumulation of allantoin in leaves, stems and roots of OsUPS1^GOS2 while in OsUPS1^RNAi allantoin was significantly accumulated in roots. We propose that OsUPS1 is responsible for allantoin partitioning in rice and its overexpression can support plant growth through accumulation of allantoin in sink tissues which can be utilized when N is limiting.     

Precision genome editing in plants via gene targeting and piggyBac‐mediated marker excision

Precise genome engineering via homologous recombination (HR)‐mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR‐mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re‐integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)‐tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants.     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

Arabidopsis thaliana ggt1 photorespiratory mutants maintain leaf carbon/nitrogen balance by reducing RuBisCO content and plant growth

Metabolic and physiological analyses of glutamate:glyoxylate aminotransferase 1 (GGT1) mutants were performed at the global leaf scale to elucidate the mechanisms involved in their photorespiratory growth phenotype. Air‐grown ggt1 mutants showed retarded growth and development, that was not observed at high CO₂ (3000 μL L^?1). When compared to wild‐type (WT) plants, air‐grown ggt1 plants exhibited glyoxylate accumulation, global changes in amino acid amounts including a decrease in serine content, lower organic acid levels, and modified ATP/ADP and NADP⁺/NADPH ratios. When compared to WT plants, their net CO₂ assimilation rates (A_n) were 50% lower and this mirrored decreases in ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) contents. High CO₂‐grown ggt1 plants transferred to air revealed a rapid decrease of A_n and photosynthetic electron transfer rate while maintaining a high energetic state. Short‐term (a night period and 4 h of light) transferred ggt1 leaves accumulated glyoxylate and exhibited low serine contents, while other amino acid levels were not modified. RuBisCO content, activity and activation state were not altered after a short‐term transfer while the ATP/ADP ratio was lowered in ggt1 rosettes. However, plant growth and RuBisCO levels were both reduced in ggt1 leaves after a long‐term (12 days) acclimation to air from high CO₂ when compared to WT plants. The data are discussed with respect to a reduced photorespiratory carbon recycling in the mutants. It is proposed that the low A_n limits nitrogen‐assimilation, this decreases leaf RuBisCO content until plants attain a new homeostatic state that maintains a constant C/N balance and leads to smaller, slower growing plants.     

Suppression of OsRAD51D results in defects in reproductive development in rice (Oryza sativa L.)

The cellular roles of RAD51 paralogs in somatic and reproductive growth have been extensively described in a wide range of animal systems and, to a lesser extent, in Arabidopsis, a dicot model plant. Here, the OsRAD51D gene was identified and characterized in rice (Oryza sativa L.), a monocot model crop. In the rice genome, three alternative OsRAD51D mRNA splicing variants, OsRAD51D.1, OsRAD51D.2, and OsRAD51D.3, were predicted. Yeast two‐hybrid studies, however, showed that only OsRAD51D.1 interacted with OsRAD51B and OsRAD51C paralogs, suggesting that OsRAD51D.1 is a functional OsRAD51D protein in rice. Loss‐of‐function osrad51d mutant rice plants displayed normal vegetative growth. However, the mutant plants were defective in reproductive growth, resulting in sterile flowers. Homozygous osrad51d mutant flowers exhibited impaired development of lemma and palea and contained unusual numbers of stamens and stigmas. During early meiosis, osrad51d pollen mother cells (PMCs) failed to form normal homologous chromosome pairings. In subsequent meiotic progression, mutant PMCs represented fragmented chromosomes. The osrad51d pollen cells contained numerous abnormal micro‐nuclei that resulted in malfunctioning pollen. The abnormalities of heterozygous mutant and T2 Ubi:RNAi‐OsRAD51D RNAi‐knock‐down transgenic plants were intermediate between those of wild type and homozygous mutant plants. The osrad51d and Ubi:RNAi‐OsRAD51D plants contained longer telomeres compared with wild type plants, indicating that OsRAD51D is a negative factor for telomere lengthening. Overall, these results suggest that OsRAD51D plays a critical role in reproductive growth in rice. This essential function of OsRAD51D is distinct from Arabidopsis, in which AtRAD51D is not an essential factor for meiosis or reproductive development.     

Co‐expression of tonoplast Cation/H+ antiporter and H+‐pyrophosphatase from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth under salinity,drought and field conditions

Salinity and drought are major environmental factors limiting the growth and productivity of alfalfa worldwide as this economically important legume forage is sensitive to these kinds of abiotic stress. In this study, transgenic alfalfa lines expressing both tonoplast NXH and H⁺‐PPase genes, ZxNHX and ZxVP1‐1 from the xerophyte Zygophyllum xanthoxylum L., were produced via Agrobacterium tumefaciens‐mediated transformation. Compared with wild‐type (WT) plants, transgenic alfalfa plants co‐expressing ZxNHX and ZxVP1‐1 grew better with greater plant height and dry mass under normal or stress conditions (NaCl or water‐deficit) in the greenhouse. The growth performance of transgenic alfalfa plants was associated with more Na⁺, K⁺ and Ca²⁺ accumulation in leaves and roots, as a result of co‐expression of ZxNHX and ZxVP1‐1. Cation accumulation contributed to maintaining intracellular ions homoeostasis and osmoregulation of plants and thus conferred higher leaf relative water content and greater photosynthesis capacity in transgenic plants compared to WT when subjected to NaCl or water‐deficit stress. Furthermore, the transgenic alfalfa co‐expressing ZxNHX and ZxVP1‐1 also grew faster than WT plants under field conditions, and most importantly, exhibited enhanced photosynthesis capacity by maintaining higher net photosynthetic rate, stomatal conductance, and water‐use efficiency than WT plants. Our results indicate that co‐expression of tonoplast NHX and H⁺‐PPase genes from a xerophyte significantly improved the growth of alfalfa, and enhanced its tolerance to high salinity and drought. This study laid a solid basis for reclaiming and restoring saline and arid marginal lands as well as improving forage yield in northern China.     

Loss of Bace2 in zebrafish affects melanocyte migration and is distinct from Bace1 knock out phenotypes

Alzheimer's disease is the most frequent dementia. Pathologically, Alzheimer's disease is characterized by the accumulation of senile plaques composed of amyloid β‐peptide (Aβ). Two proteases, β‐ and γ‐secretase proteolytically generate Aβ from its precursor, the ß‐amyloid precursor protein (APP). Inhibition of β‐secretase, also referred to as b eta‐site A PP c leaving e nzyme (BACE1) or γ‐secretase is therefore of prime interest for the development of amyloid‐lowering drugs. To assess the in vivo function of zebrafish Bace1 (zBace1), we generated zBace1 knock out fish by zinc finger nuclease‐mediated genome editing. bace1 mutants (bace1?/?) are hypomyelinated in the PNS while the CNS is not affected. Moreover, the number of mechanosensory neuromasts is elevated in bace1?/?. Mutations in zebrafish Bace2 (zBace2) revealed a distinct melanocyte migration phenotype, which is not observed in bace1?/?. Double homozygous bace1?/?; bace2?/? fish do not enhance the single mutant phenotypes indicating non‐redundant distinct physiological functions. Single homozygous bace1 mutants as well as double homozygous bace1 and bace2 mutants are viable and fertile suggesting that Bace1 is a promising drug target without major side effects. The identification of a specific bace2 ?/? associated phenotype further allows improving selective Bace1 inhibitors and to distinguish between Bace 1 and Bace 2 inhibition in vivo.

     

Agronomic nitrogen‐use efficiency of rice can be increased by driving OsNRT2.1 expression with the OsNAR2.1 promoter

The importance of the nitrate () transporter for yield and nitrogen‐use efficiency (NUE) in rice was previously demonstrated using map‐based cloning. In this study, we enhanced the expression of the OsNRT2.1 gene, which encodes a high‐affinity transporter, using a ubiquitin (Ubi) promoter and the ‐inducible promoter of the OsNAR2.1 gene to drive OsNRT2.1 expression in transgenic rice plants. Transgenic lines expressing pUbi:OsNRT2.1 or pOsNAR2.1:OsNRT2.1 constructs exhibited the increased total biomass including yields of approximately 21% and 38% compared with wild‐type (WT) plants. The agricultural NUE (ANUE) of the pUbi:OsNRT2.1 lines decreased to 83% of that of the WT plants, while the ANUE of the pOsNAR2.1:OsNRT2.1 lines increased to 128% of that of the WT plants. The dry matter transfer into grain decreased by 68% in the pUbi:OsNRT2.1 lines and increased by 46% in the pOsNAR2.1:OsNRT2.1 lines relative to the WT. The expression of OsNRT2.1 in shoot and grain showed that Ubi enhanced OsNRT2.1 expression by 7.5‐fold averagely and OsNAR2.1 promoters increased by about 80% higher than the WT. Interestingly, we found that the OsNAR2.1 was expressed higher in all the organs of pUbi:OsNRT2.1 lines; however, for pOsNAR2.1:OsNRT2.1 lines, OsNAR2.1 expression was only increased in root, leaf sheaths and internodes. We show that increased expression of OsNRT2.1, especially driven by OsNAR2.1 promoter, can improve the yield and NUE in rice.     

Oryza sativa Lysine‐Histidine‐type Transporter 1 functions in root uptake and root‐to‐shoot allocation of amino acids in rice

In agricultural soils, amino acids can represent vital nitrogen (N) sources for crop growth and yield. However, the molecular mechanisms underlying amino acid uptake and allocation are poorly understood in crop plants. This study shows that rice (Oryza sativa L.) roots can acquire aspartate at soil concentration, and that japonica subspecies take up this acidic amino acid 1.5‐fold more efficiently than indica subspecies. Genetic association analyses with 68 representative japonica or indica germplasms identified rice Lysine‐Histidine‐type Transporter 1 (OsLHT1) as a candidate gene associated with the aspartate uptake trait. When expressed in yeast, OsLHT1 supported cell growth on a broad spectrum of amino acids, and effectively transported aspartate, asparagine and glutamate. OsLHT1 is localized throughout the rice root, including root hairs, epidermis, cortex and stele, and to the leaf vasculature. Knockout of OsLHT1 in japonica resulted in reduced root uptake of amino acids. Furthermore, in ¹⁵N‐amino acid‐fed mutants versus wild‐type, a higher percentage of ¹⁵N remained in roots instead of being allocated to the shoot. ¹⁵N‐ammonium uptake and subsequently the delivery of root‐synthesized amino acids to Oslht1 shoots were also significantly decreased, which was accompanied by reduced shoot growth. These results together provide evidence that OsLHT1 functions in both root uptake and root to shoot allocation of a broad spectrum of amino acids in rice.     

Phenotypic reversion in fas mutants of Arabidopsis thaliana by reintroduction of FAS genes: variable recovery of telomeres with major spatial rearrangements and transcriptional reprogramming of 45S rDNA genes

Arabidopsis thaliana mutants dysfunctional in the evolutionarily conserved protein complex chromatin assembly factor‐1 (CAF‐1), which deposits the canonical histone H3 variant H3.1 during DNA synthesis‐dependent chromatin assembly, display complex phenotypic changes including meristem and growth alterations, sensitivity to DNA‐damaging agents, and reduced fertility. We reported previously that mutants in the FAS1 subunit of CAF‐1 progressively lose telomere and 45S rDNA repeats. Here we show that multiple aspects of the fas phenotype are recovered immediately on expression of a reintroduced FAS1 allele, and are clearly independent of the recovery of rDNA copy‐numbers and telomeres. In reverted lines, 45S rDNA genes are recovered to diverse levels with a strikingly different representation of their variants, and the typical association of nucleolar organizing region 4 with the nucleolus is perturbed. One of 45S rDNA variants (VAR1), which is silenced in wild‐type (WT) plants without mutation history (Col‐0 WT), dominates the expression pattern, whereas VAR2 is dominant in Col‐0 WT plants. We propose an explanation for the variability of telomere and 45S rDNA repeats associated with CAF‐1 function, suggesting that the differences in nuclear partitioning and expression of the rDNA variants in fas mutants and their revertants provide a useful experimental system to study genetic and epigenetic factors in gene dosage compensation.