A Rapid and Simplified Method to Isolate Specific Regulators Based on Biotin-Avidin Binding Affinities in Crops.

Transcription factors (TFs) are indispensable components of transcriptional regulatory pathways involved in crop growth and development. Herein, we developed a new method for the identification of upstream TFs specific to genes in crops based on the binding affinities of biotin and avidin. First, we constructed and verified the new biotin and avidin system (BAS) by a coprecipitation assay. Subsequently, the feasibility of DNA-based BAS (DBAS) was further proved by in vivo and in vitro assays. Furthermore, we cloned the promoter of rice OsNRT1.1B and the possible regulators were screened and identified. Additionally, partial candidates were validated by the electrophoresis mobility shift assay (EMSA), yeast one-hybrid, and luciferase activity assays. Remarkably, the results showed that the candidates PIP3 and PIP19 both responded to nitrate immediately and overexpression of PIP3 caused retard growth, which indicates that the candidates are functional and the new DBAS method is useful to isolate regulators in crops.

Genome-wide identification of nitrate-responsive microRNAs by small RNA sequencing in the rice restorer cultivar Nanhui 511.

Rice productivity relies heavily on nitrogen fertilization, and improving nitrogen use efficiency (NUE) is important for hybrid rice breeding. Reducing nitrogen inputs is the key to achieving sustainable rice production and reducing environmental problems. Here, we analyzed the genome-wide transcriptomic changes in microRNAs (miRNAs) in the indica rice restorer cultivar Nanhui 511 (NH511) under high (HN) and low nitrogen (LN) conditions. The results showed that NH511 is sensitive to nitrogen supplies and HN conditions promoted the growth its lateral roots at the seedling stage. Furthermore, we identified 483 known miRNAs and 128 novel miRNAs by small RNA sequencing in response to nitrogen in NH511. We also detected 100 differentially expressed genes (DEGs), including 75 upregulated and 25 downregulated DEGs, under HN conditions. Among these DEGs, 43 miRNAs that exhibited a 2-fold change in their expression were identified in response to HN conditions, including 28 upregulated and 15 downregulated genes. Additionally, some differentially expressed miRNAs were further validated by qPCR analysis, which showed that miR443, miR1861b, and miR166k-3p were upregulated, whereas miR395v and miR444b.1 were downregulated under HN conditions. Moreover, the degradomes of possible target genes for miR166k-3p and miR444b.1 and expression variations were analyzed by qPCR at different time points under HN conditions. Our findings revealed comprehensive expression profiles of miRNAs responsive to HN treatments in an indica rice restorer cultivar, which advances our understanding of the regulation of nitrogen signaling mediated by miRNAs and provides novel data for high-NUE hybrid rice cultivation.

Transcriptomic and Physiological Analyses of Two Rice Restorer Lines under Different Nitrogen Supplies Provide Novel Insights into Hybrid Rice Breeding.

Improving plant nitrogen-use efficiency (NUE) has great significance for various crops, particularly in hybrid breeding. Reducing nitrogen inputs is key to achieving sustainable rice production and mitigating environmental problems. In this study, we analyzed the transcriptomic and physiological changes in two indica restorer lines (Nanhui511 [NH511] and Minghui23 [MH23]) under high nitrogen (HN) and low nitrogen (LN) conditions. Compared to MH23, NH511 was more sensitive to different nitrogen supplies and exhibited higher nitrogen uptake and NUE under HN conditions by increasing lateral root and tiller numbers in the seedling and maturation stages, respectively. NH511 also exhibited a lower survival rate than MH23 when planted in a chlorate-containing hydroponic solution, indicating its HN uptake ability under different nitrogen-supply conditions. Transcriptomic analysis showed that NH511 has 2456 differentially expressed genes, whereas MH23 had only 266. Furthermore, these genes related to nitrogen utilization showed differential expression in NH511 under HN conditions, while the opposite was observed in MH23. Our findings revealed that NH511 could be regarded as elite rice and used for breeding high-NUE restorer lines by regulating and integrating nitrogen-utilization genes, which provides novel insights for the cultivation of high-NUE hybrid rice.

Field performance on grain yield and quality and genetic diversity of overwintering cultivated rice (Oryza sativa L.) in southwest China.

Understanding the field performance on grain yield and quality and the genetic diversity of overwintering (OW) cultivated rice (Oryza sativa L.) across main crop (MC) and ratooning crop (RC) is the premise to make strategies for the future OW rice variety improvement in rice production. The present field experiments were conducted in RC of 2016, in MC of both 2017 and 2018, and RC in 2019 to identify genotypes OW rice that perform stable in terms of grain yield and quality across different climate conditions. The grain yield plant-1 (GYP) and its components in six genotypes of OW rice exhibited significant difference across the 4 years (P ≤ 0.05), the maximum GYP in OW6 rice was harvested (60.28 g) in MC of 2017, but the minimum GYP in OW1 rice was harvested (33.01 g) in MC of 2018. Within six genotypes of OW rice, four grain shape traits displayed a relative small significant difference, four grain quality traits exhibited a relative small significant difference except for chalkiness rate (CR), there 226 pairs of significant PCC values between GYP and its components were calculated in all tested rice and varied from six in OW6 to eleven in OW1, there 130 pairs of significant PCC values among the four grain shape traits were calculated and ranged from twenty-one in OW1, 3, 5 to twenty-three in OW2, there 118 pairs of significant PCC values among the four grain quality traits were calculated and ranged from seventeen in OW2 to twenty-three in OW1. The numbers, directions, and size of PCC values for the grain yield and quality characters in all tested rice displayed a series of irregular variations. Six genotypes of OW rice were apparently distinguished by employing 196 pairs of simple-sequence repeats (SSRs) markers and exhibited abundant genetic diversity at the DNA level. Data from this study provide an extensive archive for the future exploration and innovation of overwintering cultivated rice variety.

Construction of three half-sib SSR linkage maps derived from overwintering cultivated rice and segregation distortion loci mapping.

Segregation distortion is a common phenomenon that has been observed in genetics and plant breeding; however, the mechanism of segregation distortion is unknown. In the present study, three half-sib F2 populations derived from three japonica overwinter (perennial) rice varieties (W1, W2, and W3) crossed to the indica rice variety Minghui725 (MH725) were developed to construct three half-sib linkage maps. We established linkage map lengths of 2032.8, 2317.4, and 2108.7 cM with average intervals of 20.1, 20.5, and 19.7 cM using 101, 113, and 107 SSR markers in W1/MH725, W2/MH725, and W3/MH725, respectively. Discrepancies in marker order and genetic linkage distance occurred in the three half-sib linkage maps due to segregation distortion. A total of 88 markers exhibited segregation distortion across the three linkage maps at P < 0.01 level, 42 segregation distortion loci (SDLs) were detected across the three half-sib populations and exhibited variable LOD value that ranged from 3.2 (SDL2f) to 30.1 (SDL5d), and 13 of the 42 SDLs were repeatedly located at the same chromosomal regions of the previously published hybrid sterility quantitative trait loci. Data from this study provide an extensive archive for investigating the genetic characteristic of overwintering cultivated rice and the future exploration and innovation of overwintering rice breeding.

Locating QTLs controlling overwintering trait in Chinese perennial Dongxiang wild rice.

Overwintering (OW) is the process by which rice passes through the winter season and germinates in the following spring. OW is also a typical quantitative inheritance trait. Currently, the molecular genetic basis of OW trait in Chinese perennial Dongxiang wild rice (DXWR) still remains to be known. In this study, a linkage map consisting of 139 simple sequence repeat (SSR) markers was constructed using an F2 population derived from a cross between DXWR and 93-11. This map covered the rice genome by approximately 1778.72 cM with approximately 12.80 cM average interval. The phenotype data of OW trait were investigated for QTL analysis in the following spring of 2017. The gene ontology (GO) annotation of the M-QTL was performed through the rice genome annotation project system. A major QTL-qOW6 was flanked by RM20069 (16,542,428 bp) and RM3498 (20,982,059 bp) on chromosome 6 and detected repeatedly by both inclusive composite interval and single-marker analysis mapping with an LOD score of 9.45 and explained 22.22% of phenotypic variance. In addition, two small QTLs (qOW2 and qOW3) controlling OW trait were detected on the second and third chromosomes, respectively. No epistatic interaction was detected between these QTLs, suggesting their unique genetic model. A total of 183 candidate genes at qOW6 locus were involved in 887 GO terms. Among them, 52 candidate genes were involved in response to stress. The other 28 candidate genes were related to cell membrane, which might affect the OW trait in perennial DXWR. These results may establish the foundation for understanding the genetic mechanism about OW trait and provide a novel gene resource for OW rice variety improvement.

Expression of Fragaria vesca PIP aquaporins in response to drought stress: PIP down-regulation correlates with the decline in substrate moisture content.

PIP aquaporin responses to drought stress can vary considerably depending on the isoform, tissue, species or level of stress; however, a general down-regulation of these genes is thought to help reduce water loss and prevent backflow of water to the drying soil. It has been suggested therefore, that it may be necessary for the plant to limit aquaporin production during drought stress, but it is unknown whether aquaporin down-regulation is gradual or triggered by a particular intensity of the stress. In this study, ten Fragaria PIP genes were identified from the woodland strawberry (Fragaria vesca L.) genome sequence and characterised at the sequence level. The water relations of F. vesca were investigated and the effect of different intensities of drought stress on the expression of four PIP genes, as well as how drought stress influences their diurnal transcription was determined. PIP down-regulation in the root corresponded to the level of drought stress. Moreover, transcript abundance of two genes highly expressed in the root (FvPIP1;1 and FvPIP2;1) was strongly correlated to the decline in substrate moisture content. The amplitude of diurnal aquaporin expression in the leaves was down-regulated by drought without altering the pattern, but showing an intensity-dependent effect. The results show that transcription of PIP aquaporins can be fine-tuned with the environment in response to declining water availability.

Analyzing lateral root development: how to move forward.

Roots are important to plants for a wide variety of processes, including nutrient and water uptake, anchoring and mechanical support, storage functions, and as the major interface between the plant and various biotic and abiotic factors in the soil environment. Therefore, understanding the development and architecture of roots holds potential for the manipulation of root traits to improve the productivity and sustainability of agricultural systems and to better understand and manage natural ecosystems. While lateral root development is a traceable process along the primary root and different stages can be found along this longitudinal axis of time and development, root system architecture is complex and difficult to quantify. Here, we comment on assays to describe lateral root phenotypes and propose ways to move forward regarding the description of root system architecture, also considering crops and the environment.

ABA signalling modulates the detection of the LM6 arabinan cell wall epitope at the surface of Arabidopsis thaliana seedling root apices.

• The hormonal and physiological regulations underpinning the cell contexts of structural features of the heterogeneous cell wall pectic polysaccharide rhamnogalacturonan-I are far from being understood. • The effect of the modulation of abscisic acid (ABA) concentrations and sensitivity on the detection of the LM6 1,5-arabinan epitope at the surface of Arabidopsis thaliana seedling root apices was assessed by means of fluorescence imaging. • Treatment with 50 nM ABA resulted in an increase in the detection of the LM6 epitope at the root surface in the region of the meristem. An inhibitor of ABA biosynthesis and introduction of the ABA synthesis mutation aba3-2 resulted in reduced epitope detection. The same ABA application resulted in an increase in the number of epidermal root meristem cells and both this and LM6 epitope detection were specifically disrupted in the abi4 ABA-insensitive mutant. These two effects were uncoupled with the application of higher ABA concentrations, which resulted in a reduction in the number of epidermal root meristem cells but increased LM6 epitope detection. • This work demonstrates a role for ABI4-mediated ABA signalling in the modulation of pectic arabinan occurrence at the A. thaliana root meristem.

Fluridone affects quiescent centre division in the Arabidopsis thaliana root stem cell niche.

Plants undergo cell division throughout their life in order to maintain their growth. It is well known that root and shoot tip of plants possess meristems, which contain quiescent cells. Fluridone (1-methyl-3-phenyl-5-(3-trifluoromethyl (phenyl))-4-(1H)-pyridinone) is an established inhibitor of both ABA and carotenoid biosynthesis. However, the other functions of fluridone remain undiscovered. In this report, we provide experimental evidence that fluridone plays a role in the division of the quiescent centre of the Arabidopsis root meristem. This study examined the effects of exogenous fluridone and ABA on the development of the stem cell niche in Arabidopsis root. We show that fluridone promoted the division of stem cells in the quiescent centre, whereas exogenous ABA suppressed quiescent centre division. Furthermore, we established a novel regulatory function for fluridone by demonstrating that it plays an important role in postembryonic development.

ABA promotes quiescence of the quiescent centre and suppresses stem cell differentiation in the Arabidopsis primary root meristem.

It is well known that abscisic acid (ABA) can halt meristems for long periods without loss of meristem function, and can also promote root growth at low concentrations, but the mechanisms underlying such regulation are largely unknown. Here we show that ABA promotes stem cell maintenance in Arabidopsis root meristems by both promoting the quiescence of the quiescent centre (QC) and suppressing the differentiation of stem cells and their daughters. We demonstrate that these two mechanisms of regulation by ABA involve distinct pathways, and identify components in each pathway. Our findings demonstrate a cellular mechanism for a positive role for ABA in promoting root meristem maintenance and root growth in Arabidopsis.

Abscisic acid is a negative regulator of root gravitropism in Arabidopsis thaliana.

The plant hormone abscisic acid (ABA) plays a role in root gravitropism and has led to an intense debate over whether ABA acts similar to auxin by translating the gravitational signal into directional root growth. While tremendous advances have been made in the past two decades in establishing the role of auxin in root gravitropism, little progress has been made in characterizing the role of ABA in this response. In fact, roots of plants that have undetectable levels of ABA and that display a normal gravitropic response have raised some serious doubts about whether ABA plays any role in root gravitropism. Here, we show strong evidence that ABA plays a role opposite to that of auxin and that it is a negative regulator of the gravitropic response of Arabidopsis roots.

Signalling mechanisms underlying the morphological responses of the root system to nitrogen in Arabidopsis thaliana.

Plants display considerable developmental plasticity in response to changing environmental conditions. The adaptations of the root system to variations in N supply are an excellent example of such developmental plasticity. In Arabidopsis, four morphological adaptations to the N supply have been characterized: (i) a localized stimulatory effect of external nitrate on lateral root elongation; (ii) a systemic inhibitory effect of high tissue nitrate concentrations on the activation of lateral root meristems; (iii) a suppression of lateral root initiation by high C:N ratios, and (iv) an inhibition of primary root growth and stimulation of root branching by external L-glutamate. These responses have provided valuable experimental systems for the study of N signalling in plants. This article will highlight some recent progress made in this direction from studies using the Arabidopsis root system. One recent development of note has been the emerging evidence of a regulatory role of nitrate transporters in some of the responses. It has been reported that the AtNRT1.1 (CHL1) dual-affinity nitrate transporter acts upstream of the ANR1 MADS box gene in mediating the stimulatory effect of a localized nitrate supply on lateral root proliferation. The AtNRT2.1 high-affinity nitrate transporter seems to be involved in the repression of lateral root initiation by high C:N ratios. The systemic inhibitory effect of high nitrate supply on lateral root development, which is mediated by abscisic acid (ABA), may be linked to the recently identified ABA receptor, FCA. The newly discovered root architectural response to external L-glutamate potentially offers a valuable experimental tool for studying the biological function of plant glutamate receptors and amino acid signalling.

A novel role for abscisic acid emerges from underground.

The continuous formation of lateral roots is a vital part of establishing a root system and enables plants to react with developmental plasticity to changing soil conditions. Evidence is accumulating that abscisic acid (ABA), which is known to be involved in stress responses, has an important role in lateral root formation. An ABA receptor mutant, fca-1, shows an altered response to ABA during lateral root formation. Interestingly, ABA seems to have distinct roles at different stages in lateral root development. The emerging role of ABA in lateral root development fits well with its general functional properties as a stress hormone, including its role in dormancy.

The hpa1 mutant of Arabidopsis reveals a crucial role of histidine homeostasis in root meristem maintenance.

Histidine (His) is an essential ingredient for protein synthesis and is required by all living organisms. In higher plants, although there is considerable evidence that His is essential for plant growth and survival, there is very little information as to whether it plays any specific role in plant development. Here, we present evidence for such a role of this amino acid in root development in Arabidopsis (Arabidopsis thaliana) from the characterization of a novel Arabidopsis mutant, hpa1, which has a very short root system and carries a mutation in one of the two Arabidopsis histidinol-phosphate aminotransferase (HPA) genes, AtHPA1. We have established that AtHPA1 encodes a functional HPA and that its complete knockout is embryo lethal. Biochemical analysis shows that the mutation in hpa1 only resulted in a 30% reduction in free His content and had no significant impact on the total His content. It did not cause any known symptoms of His starvation. However, the mutant displayed a specific developmental defect in root meristem maintenance and was unable to sustain primary root growth 2 d after germination. We have demonstrated that the root meristem failure in the mutant is tightly linked to the reduction in free His content and could be rescued by either exogenous His supplementation or AtHPA1 overexpression. Our results therefore reveal an important role of His homeostasis in plant development.

Dissecting Arabidopsis lateral root development.

Recent studies in the model plant Arabidopsis provide new insight into the regulation of root architecture, a key determinant of nutrient- and water-use efficiency in crops. Lateral root (LR) primordia originate from a subset of pericycle founder cells. Sophisticated mass-spectroscopy-based techniques have been used to map the sites of biosynthesis of auxin and its distribution in Arabidopsis seedlings, highlighting the importance of the phytohormone during LR initiation and emergence. Key components of the cell cycle and signal-transduction pathway(s) that promote and attenuate auxin-dependent LR initiation have recently been identified. Additional signals, such as abscisic acid and nitrate, also regulate LR emergence, raising intriguing questions about the cross-talk between their transduction pathways.

An abscisic acid-sensitive checkpoint in lateral root development of Arabidopsis.

Lateral root (LR) formation displays considerable plasticity in response to developmental and environmental signals. The mechanism whereby plants incorporate diverse regulatory signals into the developmental programme of LRs remains to be elucidated. Current concepts of lateral root regulation focus on the role of auxin. In this study, we show that another plant hormone, abscisic acid (ABA), also plays a critical role in the regulation of this post-embryonic developmental event. In the presence of exogenous ABA, LR development is inhibited. This occurs at a specific developmental stage, i.e. immediately after the emergence of the LR primordium (LRP) from the parent root and prior to the activation of the LR meristem, and is reversible. Interestingly, this inhibition requires 10-fold less ABA than the inhibition of seed germination and is only slightly reduced in characterised abi mutants, suggesting that it may involve novel ABA signalling mechanisms. We also present several lines of evidence to support the conclusion that the ABA-induced lateral root inhibition is mediated by an auxin-independent pathway. First, the inhibition could not be rescued by either exogenous auxin application or elevated auxin synthesis. Secondly, a mutation in the ALF3 gene, which is believed to encode an important component in the auxin-dependent regulatory pathway for the post-emergence LR development, does not affect the sensitivity of LRs to ABA. Thirdly, ABA and the alf3-1 mutation do not act at the same developmental point. To summarise, these results demonstrate a novel ABA-sensitive, auxin-independent checkpoint for lateral root development in Arabidopsis at the post-emergence stage. In addition, we also present data indicating that regulation of this developmental checkpoint may require novel ABA signalling mechanisms and that ABA suppresses auxin response in the LRPs.