Overexpression of oilseed rape trehalose-6-phosphate synthesis gene BnaC02.TPS8 confers sensitivity to low nitrogen and high sucrose-induced anthocyanin accumulation in Arabidopsis.

MAIN CONCLUSION: Overexpression of BnaC02.TPS8 increased low N and high sucrose-induced anthocyanin accumulation. Anthocyanin plays a crucial role in safeguarding photosynthetic tissues against high light, UV radiation, and oxidative stress. Their accumulation is triggered by low nitrogen (N) stress and elevated sucrose levels in Arabidopsis. Trehalose-6-phosphate (T6P) serves as a pivotal signaling molecule, sensing sucrose availability, and carbon (C) metabolism. However, the mechanisms governing the regulation of T6P synthase (TPS) genes responsible for anthocyanin accumulation under conditions of low N and high sucrose remain elusive. In a previous study, we demonstrated the positive impact of a cytoplasm-localized class II TPS protein 'BnaC02.TPS8' on photosynthesis and seed yield improvement in Brassica napus. The present research delves into the biological role of BnaC02.TPS8 in response to low N and high sucrose. Ectopic overexpression of BnaC02.TPS8 in Arabidopsis seedlings resulted in elevated shoot T6P levels under N-sufficient conditions, as well as an increased carbon-to-nitrogen (C/N) ratio, sucrose accumulation, and starch storage under low N conditions. Overexpression of BnaC02.TPS8 in Arabidopsis heightened sensitivity to low N stress and high sucrose levels, accompanied by increased anthocyanin accumulation and upregulation of genes involved in flavonoid biosynthesis and regulation. Metabolic profiling revealed increased levels of intermediate products of carbon metabolism, as well as anthocyanin and flavonoid derivatives in BnaC02.TPS8-overexpressing Arabidopsis plants under low N conditions. Furthermore, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) analyses demonstrated that BnaC02.TPS8 interacts with both BnaC08.TPS9 and BnaA01.TPS10. These findings contribute to our understanding of how TPS8-mediated anthocyanin accumulation is modulated under low N and high sucrose conditions.

Rice transcription factor OsWRKY37 positively regulates flowering time and grain fertility under copper deficiency.

Efficient uptake, translocation, and distribution of Cu to rice (Oryza sativa) spikelets is crucial for flowering and yield production. However, the regulatory factors involved in this process remain unidentified. In this study, we isolated a WRKY transcription factor gene induced by Cu deficiency, OsWRKY37, and characterized its regulatory role in Cu uptake and transport in rice. OsWRKY37 was highly expressed in rice roots, nodes, leaf vascular bundles, and anthers. Overexpression of OsWRKY37 promoted the uptake and root-to-shoot translocation of Cu in rice under -Cu condition but not under +Cu condition. While mutation of OsWRKY37 significantly decreased Cu concentrations in the stamen, the root-to-shoot translocation and distribution ratio in brown rice affected pollen development, delayed flowering time, decreased fertility, and reduced grain yield under -Cu condition. yeast one-hybrid, transient co-expression and EMSAs, together with in situ RT-PCR and RT-qPCR analysis, showed that OsWRKY37 could directly bind to the upstream promoter region of OsCOPT6 (copper transporter) and OsYSL16 (yellow stripe-like protein) and positively activate their expression levels. Analyses of oscopt6 mutants further validated its important role in Cu uptake in rice. Our study demonstrated that OsWRKY37 acts as a positive regulator involved in the uptake, root-to-shoot translocation, and distribution of Cu through activating the expression of OsCOPT6 and OsYSL16, which is important for pollen development, flowering, fertility, and grain yield in rice under Cu deficient conditions. Our results provide a genetic strategy for improving rice yield under Cu deficient condition.

Proteomics reveals the significance of vacuole Pi transporter in the adaptability of Brassica napus to Pi deprivation.

Vacuolar Pi transporters (VPTs) have recently been identified as important regulators of cellular Pi status in Arabidopsis thaliana and Oryza sativa. In the oil crop Brassica napus, BnA09PHT5;1a and BnC09PHT5;1a are two homologs of AtPHT5;1, the vacuolar Pi influx transporter in Arabidopsis. Here, we show that Pi deficiency induces the transcription of both homologs of PHT5;1a genes in B. napus leaves. Brassica PHT5;1a double mutants (DM) had smaller shoots and higher cellular Pi concentrations than wild-type (WT, Westar 10), suggesting the potential role of BnPHT5;1a in modulating cellular Pi status in B. napus. A proteomic analysis was performed to estimate the role of BnPHT5;1a in Pi fluctuation. Results show that Pi deprivation disturbs the abundance of proteins in the physiological processes involved in carbohydrate metabolism, response to stimulus and stress in B. napus, while disruption of BnPHT5;1a genes may exacerbate these processes. Besides, the processes of cell redox homeostasis, lipid metabolic and proton transmembrane transport are supposed to be unbalanced in BnPHT5;1a DM under the -Pi condition. Noteworthy, disruption of BnPHT5;1a genes severely alters the abundance of proteins related to ATP biosynthesis, and proton/inorganic cation transmembrane under normal Pi condition, which might contribute to B. napus growth limitations. Additionally, seven new protein markers of Pi homeostasis are identified in B. napus. Taken together, this study characterizes the important regulatory role of BnPHT5;1a genes as vacuolar Pi influx transporters in Pi homeostasis in B. napus.

BnaA4.BOR2 contributes the tolerance of rapeseed to boron deficiency by improving the transport of boron from root to shoot.

Boron (B) is essential for plant growth. However, the molecular mechanism of B transport in rapeseed (Brassica napus L.) is unknown well. Here, we report that B transporter BnaA4.BOR2 is involved in the transport of B from root to shoot and its distribution in shoot cell wall and flower in rapeseed. The results of GUS staining and in-situ PCR analysis showed that BnaA4.BOR2 is mainly expressed in cortex and endodermis of root tip meristem zone and endodermis of mature zone. BnaA4.BOR2 was mainly localized in plasma membrane and showed B transport activity in yeast. Overexpression of Bna4.BOR2 could rescue the phenotype of Arabidopsis mutant bor2-2 under low-B condition. Furthermore, knockout of BnaA4.BOR2 could significantly enhance the sensitivity of rapeseed mutants to B deficiency, including inhibition of root elongation and biomass decrease of roots and shoots. The B concentration in xylem sap of BnaA4.BOR2 mutants was significantly decreased under B deficiency, which resulted in significantly lower B concentrations in shoot cell wall at seedling stage and flower organ at reproductive stage compared to that of wild-type QY10. The growth of BnaA4.BOR2 mutants were severely inhibited, exhibiting a typical B-deficient phenotype of "flowering without seed setting", leading to a sharp decrease in seed yield in B deficient soil. Taken together, these results indicate that BnaA4.BOR2 is critical for rapeseed growth and seed yield production under low B level, which is mainly expressed in cortex and endodermis, and contributed to the transport of B from roots to shoots and its distribution in shoot.

Trade-offs between root-secreted acid phosphatase and root morphology traits, and their contribution to phosphorus acquisition in Brassica napus.

Oilseed rape (Brassica napus) is one of the most important oil crops in the world and shows sensitivity to low phosphorus (P) availability. In many soils, organic P (Po) is the main component of the soil P pool. Po must be mineralised to Pi through phosphatases, and then taken up by plants. However, the relationship between root-secreted acid phosphatases (APase) and root morphology traits, two important P-acquisition strategies in response to P deficiency, is unclear among B. napus genotypes. This study aimed to understand their relationship and how they affect P acquisition, which is crucial for the sustainable utilisation of agricultural P resources. This study showed significant genotypic variations in root-secreted APase activity per unit root fresh weight (SAP) and total root-secreted APase activity per plant (total SAP) among 350 B. napus genotypes. Seed yield was positively correlated with total SAP but not significantly correlated with SAP. Six root traits of 18 B. napus genotypes with contrasting root biomass were compared under normal Pi, low Pi and Po. Genotypes with longer total root length (TRL) reduced SAP, but those with shorter TRL increased SAP under P deficiency. Additionally, TRL was important in P-acquisition under three P treatments, and total SAP was also important in P-acquisition under Po treatment. In conclusion, trade-offs existed between the two P-acquisition strategies among B. napus genotypes under P-deficient conditions. Total SAP was an important root trait under Po conditions. These results might help to breed B. napus with greater P-acquisition ability under low P availability conditions.

Arabidopsis transcription factor STOP1 directly activates expression of NOD26-LIKE MAJOR INTRINSIC PROTEIN5;1, and is involved in the regulation of tolerance to low-boron stress.

Transcriptional regulation is a crucial component of plant adaptation to numerous different stresses; however, its role in how plants adapt to low-boron (B) stress remains unclear. In this study, we show that the C2H2-type transcription factor SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) in Arabidopsis is essential for improving plant growth under low-B conditions. STOP1 and the boric acid-channel protein NOD26-LIKE MAJOR INTRINSIC PROTEIN5;1 (NIP5;1) were found to co-localize in root epidermal cells, and STOP1 binds to the 5´-untranslated region of NIP5;1 to activate its expression and enhance B uptake by the roots. Overexpression of STOP1 increased tolerance to low-B stress by up-regulating NIP5;1 transcript levels. Further genetic analyses revealed that STOP1 and NIP5;1 function together in the same pathway to confer low-B tolerance. These results highlight the importance of the STOP1-NIP5;1 module in improving plant growth under low-B conditions.

Identification and function characterization of BnaBOR4 genes reveal their potential for Brassica napus cultivation under high boron stress.

Boron (B) is essential for plant growth, but toxic in excess. In several countries, soil toxic B levels are always a severe agricultural problem in arid and semi-arid regions. Phytoremediation of excess B containing soil is still in its infancy, while high B tolerant plants with elevated protein abundance of B efflux transporter were successfully established or explored. Brassica napus (B. napus) is one of the most important oil crops. However, B efflux transporters underlying excess B tolerance in B. napus remain unknown. Here, we reported that in Brassicaceae species, B. napus had four homologous genes of Arabidopsis AtBOR4 , which were renamed BnaBOR4.1, BnaBOR4.2, BnaBOR4.3 and BnaBOR4.4. BnaBOR4.1, BnaBOR4.2 and BnaBOR4.3 showed constitutive expression and BnaBOR4.4 appeared to be a pseudogene. BnaBOR4.2 and BnaBOR4.3 were expressed in inner cell layers and BnaBOR4.1 in the outer cell layer in root tip, and all were expressed in vascular tissue in the mature zone. B efflux activity assays in yeast demonstrated that BnaBOR4.1, BnaBOR4.2 and AtBOR4 but not BnaBOR4.3 had comparable levels of B transport activity. Structure-functional analysis between BnaBOR4.3 and BnaBOR4.2 demonstrated that amino acid residue substitution at position 297 (Ala vs Pro) and 427 (Met vs Leu) is critical for the B transport activity. Mutant BnaBOR4.3M427L partially restored the B efflux activity, and both mutants BnaBOR4.3A297P and BnaBOR4.3A297P&M427L fully restored B efflux activity, indicating that the Pro297 residue is critical for their function. Further validation of BnaBOR4 was accomplished by growing transgenic Arabidopsis plants under high B conditions. Taken together, our study identified two functional B efflux genes BnaBOR4.1 and BnaBOR4.2 in B. napus, and a key amino acid residue proline 297 associated with B efflux activity. This study highlights the potential of BanBOR4 genes for B. napus cultivation under high B stress.

Trehalose-6-phosphate synthase 8 increases photosynthesis and seed yield in Brassica napus.

Trehalose-6-phosphate (T6P) functions as a vital proxy for assessing carbohydrate status in plants. While class II T6P synthases (TPS) do not exhibit TPS activity, they are believed to play pivotal regulatory roles in trehalose metabolism. However, their precise functions in carbon metabolism and crop yield have remained largely unknown. Here, BnaC02.TPS8, a class II TPS gene, is shown to be specifically expressed in mature leaves and the developing pod walls of Brassica napus. Overexpression of BnaC02.TPS8 increased photosynthesis and the accumulation of sugars, starch, and biomass compared to wild type. Metabolomic analysis of BnaC02.TPS8 overexpressing lines and CRISPR/Cas9 mutants indicated that BnaC02.TPS8 enhanced the partitioning of photoassimilate into starch and sucrose, as opposed to glycolytic intermediates and organic acids, which might be associated with TPS activity. Furthermore, the overexpression of BnaC02.TPS8 not only increased seed yield but also enhanced seed oil accumulation and improved the oil fatty acid composition in B. napus under both high nitrogen (N) and low N conditions in the field. These results highlight the role of class II TPS in impacting photosynthesis and seed yield of B. napus, and BnaC02.TPS8 emerges as a promising target for improving B. napus seed yield.

Nitrate alleviates ammonium toxicity in Brassica napus by coordinating rhizosphere and cell pH and ammonium assimilation.

In natural and agricultural situations, ammonium ( NH 4 + ) is a preferred nitrogen (N) source for plants, but excessive amounts can be hazardous to them, known as NH 4 + toxicity. Nitrate ( NO 3 - ) has long been recognized to reduce NH 4 + toxicity. However, little is known about Brassica napus, a major oil crop that is sensitive to high NH 4 + . Here, we found that NO 3 - can mitigate NH 4 + toxicity by balancing rhizosphere and intracellular pH and accelerating ammonium assimilation in B. napus. NO 3 - increased the uptake of NO 3 - and NH 4 + under high NH 4 + circumstances by triggering the expression of NO 3 - and NH 4 + transporters, while NO 3 - and H+ efflux from the cytoplasm to the apoplast was enhanced by promoting the expression of NO 3 - efflux transporters and genes encoding plasma membrane H+ -ATPase. In addition, NO 3 - increased pH in the cytosol, vacuole, and rhizosphere, and down-regulated genes induced by acid stress. Root glutamine synthetase (GS) activity was elevated by NO 3 - under high NH 4 + conditions to enhance the assimilation of NH 4 + into amino acids, thereby reducing NH 4 + accumulation and translocation to shoot in rapeseed. In addition, root GS activity was highly dependent on the environmental pH. NO 3 - might induce metabolites involved in amino acid biosynthesis and malate metabolism in the tricarboxylic acid cycle, and inhibit phenylpropanoid metabolism to mitigate NH 4 + toxicity. Collectively, our results indicate that NO 3 - balances both rhizosphere and intracellular pH via effective NO 3 - transmembrane cycling, accelerates NH 4 + assimilation, and up-regulates malate metabolism to mitigate NH 4 + toxicity in oilseed rape.

Phosphate Transporter BnaPT37 Regulates Phosphate Homeostasis in Brassica napus by Changing Its Translocation and Distribution In Vivo.

Inorganic phosphate (Pi) is actively taken up by Pi transporters (PTs) from the soil and transported into the plant. Here, we functionally characterized the Brassica napus gene BnaPT37, which belongs to the PHT1 family. BnaPT37 is a plasma membrane-localized protein containing 534 amino acids. Expression of BnaPT37 increased significantly under Pi deficiency in various tissues, especially in fully expanded leaves. Expression of the ß-glucuronidase reporter gene driven by the BnaPT37 promoter showed that BnaPT37 is expressed in the root, stem, calyx, and leaf under Pi deficiency. BnaPT37 can complement a yeast mutant strain defective in five Pi transporters and can restore the growth of the Arabidopsis atpt1/2 double mutant under Pi deprivation. Overexpression of BnaPT37 in rapeseed significantly increased Pi translocation from root to shoot. Moreover, the movement of Pi from fully expanded leaves to new leaves and roots was enhanced in the transgenic lines compared to the wild type. However, the overexpression of BnaPT37 inhibited the flowering time, plant height, and Pi accumulation in seeds. In conclusion, BnaPT37 functions as a plasma membrane-localized Pi transporter and might be involved in Pi translocation from root to shoot and Pi distribution from source to sink in B. napus.

A critical review of plant adaptation to environmental boron stress: Uptake, utilization, and interplay with other abiotic and biotic factors.

Boron (B) is an indispensable mineral nutrient for plants and is primarily taken up by roots mainly in the form of boric acid (H3BO3). Recently, research shows that B has a significant impact on plant growth and productivity due to its narrow range between deficiency and toxicity. Fertilization and other procedures to address B stress (deficiency and toxicity) in soils are generally expensive and time-consuming. Over the past 20 years, substantial studies have been conducted to investigate the mechanisms underlying B acquisition and the molecular regulation of B stress in plants. In this review, we discuss the effects of B stress on plant growth, physiology, and biochemistry, and finding on enhancing plant tolerance from the perspective of plant B uptake, transport, and utilization. We also refer to recent results demonstrating the interactions among B and other biological and abiotic factors, including nitrogen, phosphorus, aluminum, and microorganisms. Finally, emerging trends in this field are discussed.

Optimal phosphorus management strategies to enhance crop productivity and soil phosphorus fertility in rapeseed-rice rotation.

Optimal phosphorus (P) managements can improve the crop yield without reducing soil P supply capacity over the long term. In this study, the rapeseed-rice rotation experiments were conducted to evaluate the effect of five optimal P fertilizer managements, including the addition of RA (rooting agents), PSB (phosphate solubilizing bacteria), CMP (calcium and magnesium phosphate fertilizer), DP1 (starter P) and DP2 (foliar fertilizer) with the reduction of 40% (in the 1st rapeseed season) and 75% (in the 2nd rapeseed season) P fertilizers of farmers' fertilizer practice (FFP) on crop productivity and soil P fertility in low and high P fertility soils. Seed yield, P partial factor productivity, and P recovery efficiency of both cultivars, Shengguang168 (SG168) and Zhongshuang 11 (ZS11), were significantly improved under optimal P managements, and the increase of them in low P fertility soil was more than that in high P fertility soil. Total P surplus was lower under optimal P managements than under FFP in both P fertility soils. The increasing amount of crop yields under optimal P managements for both cultivars was equivalent to that of 16.0-38.3 kg P2O5 hm-2 of P fertilizer application, and the order of the optimal P managements was as follows: RA > PSB > CMP > DP1 > DP2. In addition, the grain yield of rotated rice cultivar Longliangyou1212 (LLY1212) without P supply was not reduced in both fertility soils. Compared with low P fertility soil, yields of SG168, ZS11 and LLY1212 in high P fertility soil increased by 28.1%-71.7%, 28.3%-78.9% and 26.2%-47.2% at the same treatment, respectively. In summary, optimal P managements in the rapeseed season could stabilize the crop yield, promote P use efficiency and the capacity of soil P supply in the rapeseed-rice rotation, especially in low P fertility soil.

The transcription factor BnaA9.WRKY47 coordinates leaf senescence and nitrogen remobilization in Brassica napus.

Nitrogen (N) is an essential macronutrient for plants, and its remobilization is key for adaptation to deficiency stress. However, there is limited understanding of the regulatory mechanisms of N remobilization in the important crop species Brassica napus (oilseed rape). Here, we report the identification of a transcription factor, BnaA9.WRKY47, that is induced by N starvation in a canola variety. At the seedling stage, BnaA9.WRKY47-overexpressing (OE) lines displayed earlier senescence of older leaves and preferential growth of juvenile leaves compared to the wild type under N starvation. At the field scale, the seed yield was significantly increased in the BnaA9.WRKY47-OE lines compared with the wild type when grown under N deficiency conditions and, conversely, it was reduced in BnaA9.WRKY47-knockout mutants. Biochemical analyses demonstrated that BnaA9.WRKY47 directly activates BnaC7.SGR1 to accelerate senescence of older leaves. In line with leaf senescence, the concentration of amino acids in the older leaves of the OE lines was elevated, and the proportion of plant N that they contained was reduced. This was associated with BnaA9.WRKY47 activating the amino acid permease BnaA9.AAP1 and the nitrate transporter BnaA2.NRT1.7. Thus, the expression of BnaA9.WRKY47 efficiently facilitated N remobilization from older to younger leaves or to seeds. Taken together, our results demonstrate that BnaA9.WRKY47 up-regulates the expression of BnaC7.SGR1, BnaA2.NRT1.7, and BnaA9AAP1, thus promoting the remobilization of N in B. napus under starvation conditions.

Integrating genome-wide association studies with selective sweep reveals genetic loci associated with tolerance to low phosphate availability in Brassica napus.

Oilseed rape (Brassica napus L.; B. napus) is an important oil crop worldwide. However, the genetic mechanisms of B. napus adaptations to low phosphate (P) stress are largely unknown. In this study, a genome-wide association study (GWAS) identified 68 SNPs significantly associated with seed yield (SY) under low P (LP) availability, and 7 SNPs significantly associated with phosphorus efficiency coefficient (PEC) in two trials. Among these SNPs, two, chrC07__39807169 and chrC09__14194798, were co-detected in two trials, and BnaC07.ARF9 and BnaC09.PHT1;2 were identified as candidate genes of them, respectively, by combining GWAS with quantitative reverse-transcription PCR (qRT-PCR). There were significant differences in the gene expression level of BnaC07.ARF9 and BnaC09.PHT1;2 between P-efficient and -inefficiency varieties at LP. SY_LP had a significant positive correlation with the gene expression level of both BnaC07.ARF9 and BnaC09.PHT1;2. BnaC07.ARF9 and BnaA01.PHR1 could directly bind the promoters of BnaA01.PHR1 and BnaC09.PHT1;2, respectively. Selective sweep analysis was conducted between ancient and derived B. napus, and detected 1280 putative selective signals. Within the selected region, a large number of genes related to P uptake, transport, and utilization were detected, such as purple acid phosphatase (PAP) family genes and phosphate transporter (PHT) family genes. These findings provide novel insights into the molecular targets for breeding P efficiency varieties in B. napus. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01399-9.

The overexpression of LOW PHOSPHATE ROOT 1 (LPR1) negatively regulates Arabidopsis growth in response to Cadmium (Cd) stress.

Cadmium (Cd) is a highly toxic element that is easily absorbed by plant, and the mechanisms of the plant response to Cd toxicity are very complex. In this study, the role of LPR1 (LOW Phosphate Root 1) encoding a cell-wall-targeted ferroxidase in Cd stress was investigated. The results showed that the overexpression of LPR1 caused an average reduction of 23%-40% in the primary root lengths, 67%-73% in the fresh weights, 32%-46% in the lengths of the non-root hair zone (NRHZ) and 70%-71% in the chlorophyll contents in both LPR1-OX lines when compared with the wild type (WT), while there were no significant changes in these traits between the WT and mutant lpr1 lines under Cd stress (7.5 µmol/L CdSO4). Further investigation showed that the overexpression of LPR1 triggered reactive oxygen species (ROS) bursts and reduced the entry of available iron (Fe2+) into the cell, which induced the expression of iron-regulated transporter 1 (IRT1). The up-regulation of IRT1 contributed to the increase of Cd accumulation and growth retardation under Cd stress. Exogenous Fe and ROS scavengers down-regulated the IRT1's expression and alleviated the growth inhibition in LPR1-OX lines, indicating that LPR1-dependent ROS up-regulated IRT1, which subsequently exacerbated the Cd influx into plants. Our findings highlight a pathway of LPR1-mediated plant responding to Cd toxicity stress through the regulation of ROS and Fe homeostasis.

Metabolomic analysis reveals key metabolites alleviating green spots under exogenous sucrose spraying in air-curing cigar tobacco leaves.

Cigar variety CX-010 tobacco leaves produce localized green spots during the air-curing period, and spraying exogenous sucrose effectively alleviates the occurrence of the green spots. To investigate the alleviation effect of exogenous sucrose spraying, the total water content and the number and size of green spots on tobacco leaves were investigated during the air-curing period under four treatments; CK (pure water), T1 (0.1 M sucrose), T2 (0.2 M sucrose) and T3 (0.4 M sucrose). The results showed that the total water content of tobacco leaves showed a trend of T3 < CK < T2 < T1 in the early air-curing stage, and the number and size of green spots showed a trend of T3 < T2 < T1 < CK. All sucrose treatments alleviated the green spot phenomenon, and T3 had the fewest green spots. Thus, the tobacco leaves of the T3 and CK treatments at two air-curing stages were used to perform metabolomics analysis with nontargeted liquid chromatography‒mass spectrometry to determine the physiological mechanism. A total of 259 and 178 differentially abundant metabolites (DAMs) between T3- and CK-treated tobacco leaves were identified in the early air-curing and the end of air-curing stages, respectively. These DAMs mainly included lipid and lipid-like molecules, carbohydrates, and organic acids and their derivatives. Based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, the T3 treatment significantly altered carbohydrate metabolism (pentose phosphate pathway, sucrose and starch metabolism and galactose metabolism) and amino acid metabolism (tyrosine metabolism and tryptophan metabolism) in air-curing tobacco leaves. Sucrose treatment alleviated green spots by altering DAMs that affected chlorophyll degradation, such as tyrosine and citric acid, to promote the normal degradation of chlorophyll.

Brassinosteroid signaling regulates phosphate starvation-induced malate secretion in plants.

Inorganic phosphate (Pi) is often limited in soils due to precipitation with iron (Fe) and aluminum (Al). To scavenge heterogeneously distributed phosphorus (P) resources, plants have evolved a local Pi signaling pathway that induces malate secretion to solubilize the occluded Fe-P or Al-P oxides. In this study, we show that Pi limitation impaired brassinosteroid signaling and downregulated BRASSINAZOLE-RESISTANT 1 (BZR1) expression in Arabidopsis thaliana. Exogenous 2,4-epibrassinolide treatment or constitutive activation of BZR1 (in the bzr1-D mutant) significantly reduced primary root growth inhibition under Pi-starvation conditions by downregulating ALUMINUM-ACTIVATED MALATE TRANSPORTER 1 (ALMT1) expression and malate secretion. Furthermore, AtBZR1 competitively suppressed the activator effect of SENSITIVITY TO PROTON RHIZOTOXICITY 1 (STOP1) on ALMT1 expression and malate secretion in Nicotiana benthamiana leaves and Arabidopsis. The ratio of nuclear-localized STOP1 and BZR1 determined ALMT1 expression and malate secretion in Arabidopsis. In addition, BZR1-inhibited malate secretion is conserved in rice (Oryza sativa). Our findings provide insight into plant mechanisms for optimizing the secretion of malate, an important carbon resource, to adapt to Pi-deficiency stress.

Rapid identification of a major locus qPRL-C06 affecting primary root length in Brassica napus by QTL-seq.

BACKGROUND AND AIMS: Brassica napus is one of the most important oilseed crops worldwide. Seed yield of B. napus significantly correlates with the primary root length (PRL). The aims of this study were to identify quantitative trait loci (QTLs) for PRL in B. napus. METHODS: QTL-seq and conventional QTL mapping were jointly used to detect QTLs associated with PRL in a B. napus double haploid (DH) population derived from a cross between 'Tapidor' and 'Ningyou 7'. The identified major locus was confirmed and resolved by an association panel of B. napus and an advanced backcross population. RNA-seq analysis of two long-PRL lines (Tapidor and TN20) and two short-PRL lines (Ningyou 7 and TN77) was performed to identify differentially expressed genes in the primary root underlying the target QTLs. KEY RESULTS: A total of 20 QTLs impacting PRL in B. napus grown at a low phosphorus (P) supply were found by QTL-seq. Eight out of ten QTLs affecting PRL at a low P supply discovered by conventional QTL mapping could be detected by QTL-seq. The locus qPRL-C06 identified by QTL-seq was repeatedly detected at both an optimal P supply and a low P supply by conventional QTL mapping. This major constitutive QTL was further confirmed by regional association mapping. qPRL-C06 was delimited to a 0.77 Mb genomic region on chromosome C06 using an advanced backcross population. A total of 36 candidate genes within qPRL-C06 were identified that showed variations in coding sequences and/or exhibited significant differences in mRNA abundances in primary root between the long-PRL and short-PRL lines, including five genes involved in phytohormone biosynthesis and signaling. CONCLUSIONS: These results both demonstrate the power of the QTL-seq in rapid QTL detection for root traits and will contribute to marker-assisted selective breeding of B. napus cultivars with increased PRL.

Physiological Responses of Cigar Tobacco Crop to Nitrogen Deficiency and Genome-Wide Characterization of the NtNPF Family Genes.

Tobacco prefers nitrate as a nitrogen (N) source. However, little is known about the molecular components responsible for nitrate uptake and the physiological responses of cigar tobacco to N deficiency. In this study, a total of 117 nitrate transporter 1 (NRT1) and peptide transporter (PTR) family (NPF) genes were comprehensively identified and systematically characterized in the whole tobacco genome. The NtNPF members showed significant genetic diversity within and across subfamilies but showed conservation between subfamilies. The NtNPF genes are dispersed unevenly across the chromosomes. The phylogenetic analysis revealed that eight subfamilies of NtNPF genes are tightly grouped with their orthologues in Arabidopsis. The promoter regions of the NtNPF genes had extensive cis-regulatory elements. Twelve core NtNPF genes, which were strongly induced by N limitation, were identified based on the RNA-seq data. Furthermore, N deprivation severely impaired plant growth of two cigar tobaccos, and CX26 may be more sensitive to N deficiency than CX14. Moreover, 12 hub genes respond differently to N deficiency between the two cultivars, indicating the vital roles in regulating N uptake and transport in cigar tobacco. The findings here contribute towards a better knowledge of the NtNPF genes and lay the foundation for further functional analysis of cigar tobacco.

Identification of vacuolar phosphate influx transporters in Brassica napus.

Recent progress has shown that vacuolar Pi transporters (VPTs) are important for cellular Pi homoeostasis in Arabidopsis thaliana and Oryza sativa under fluctuating external Pi supply, but the identity and involvement of VPTs in cellular Pi homoeostasis in Brassica napus is poorly understood. Here, we identified two vacuolar Pi influx transporters B. napus, BnA09PHT5;1b and BnCnPHT5;1b, and uncovered their necessity for cellular Pi homoeostasis through functional analysis. Both Brassica proteins are homologs of Arabidopsis AtPHT5;1 with a similar sequence, structure, tonoplast localization, and VPT activity. Brassica pht5;1b double mutants had smaller shoots and larger shoot cellular Pi concentrations than wild-type B. napus, which contrasts with a previous study of the Arabidopsis pht5;1 mutant, suggesting that PHT5;1-VPTs play different roles in cellular Pi homoeostasis in seedlings of B. napus and A. thaliana. Disruption of BnPHT5;1b genes also caused Pi toxicity in floral organs, reduced seed yield and impacted seed traits, consistent with the proposed role of AtPHT5;1 in floral Pi homoeostasis in Arabidopsis. Taken together, our studies identified two vacuolar Pi influx transporters in B. napus and revealed the distinct and conserved roles of BnPHT5;1bs in cellular Pi homoeostasis in this plant species.