A Review: Systemic Signaling in the Regulation of Plant Responses to Low N, P and Fe.

Plant signal transduction occurs in response to nutrient element deficiency in plant vascular tissue. Recent works have shown that the vascular tissue is a central regulator in plant growth and development by transporting both essential nutritional and long-distance signaling molecules between different parts of the plant's tissues. Split-root and grafting studies have deciphered the importance of plants' shoots in receiving root-derived nutrient starvation signals from the roots. This review assesses recent studies about vascular tissue, integrating local and systemic long-distance signal transduction and the physiological regulation center. A substantial number of studies have shown that the vascular tissue is a key component of root-derived signal transduction networks and is a regulative center involved in plant elementary nutritional deficiency, including nitrogen (N), phosphate (P), and iron (Fe).

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain.

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species. Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium. The plants were then acclimated to a soil environment and tested for the presence of a transgene using the ß-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.

Overexpression of Nicotianamine Synthase (AtNAS1) Increases Iron Accumulation in the Tuber of Potato.

Iron (Fe) deficiency is a global health problem, especially in underdeveloped countries. Biofortification with genetic engineering methods has been used to improve Fe nutrition in a number of crops. Various steps, e.g., uptake, distribution, and storage, involved in Fe homeostasis have been manipulated to increase the Fe concentration in the edible portions of plants. Nicotianamine (NA) is an important metal ion chelator in plants. It promotes the mobility of Fe and decreases cellular Fe toxicity. Increasing the Fe content in crops by promoting NA synthesis could help decrease human diseases associated with Fe deficiency. In the present study, Arabidopsis thaliana nicotianamine synthase 1 (AtNAS1) was overexpressed in potato (Solanum tuberosum, St) under the control of the cauliflower mosaic virus 35S promoter. Transgenic plants had a significantly increased amount of Fe in tubers (52.7 µg/g dry weight, 2.4-fold the amount in wild-type tubers), while no differences in plant phenotype or yield were detected between transgenic and wild-type plants. The expression of genes involved in root mineral uptake and homeostasis, such as StYSL1, StIRT1, StFRO1, and StNAS, was also altered in the roots and leaves of the transgenic plants. Our results demonstrate that the manipulation of Fe chelation is a useful strategy for Fe nutrition improvement, and the increased Fe accumulation in tubers of transgenic potato plants is most likely caused by the increased movement of Fe from the leaf to the tuber.

Functional study and elite haplotype identification of soybean phosphate starvation response transcription factors GmPHR14 and GmPHR32.

Phosphorus (P) is one of the important mineral elements required for plant growth and development. However, because of the low mobility in soil, P deficiency has been an important factor limiting soybean production. Here, we identified 14 PHR (phosphate starvation response) genes in soybean genome and verified that two previously unreported GmPHR members, GmPHR14 and GmPHR32, were involved in low-P stress tolerance in soybean. GmPHR14 and GmPHR32 were present in two diverged branches of the phylogenic tree. Both genes were highly expressed in roots and root nodules and were induced by P deficiency. GmPHR14 and GmPHR32 both were expressed in the nucleus. The 211 amino acids in the N terminus of GmPHR32 were found to be required for the transcriptional activity. Overexpressing GmPHR14 or GmPHR32 in soybean hairy roots significantly increased roots and shoots dry weight under low-P condition, and overexpressing GmPHR14 additionally significantly increased roots P concentration under low-P condition. GmPHR14 and GmPHR32 were polymorphic in soybean population and the elite haplotype2 (Hap2) for both genes was preferentially present in improved cultivars and showed significantly higher shoots dry weight under low-P condition than the other two haplotypes. These results suggested GmPHR14 and GmPHR32 both positively regulated low-P responses in soybean, and would shed light on the molecular mechanism of low-P stress tolerance. Furthermore, the identified elite haplotypes would be useful in P-efficient soybean breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01301-z.

RBP differentiation contributes to selective transmissibility of OPT3 mRNAs.

Long-distance mobile mRNAs play key roles in gene regulatory networks that control plant development and stress tolerance. However, the mechanisms underlying species-specific delivery of mRNA still need to be elucidated. Here, the use of grafts involving highly heterozygous apple (Malus) genotypes allowed us to demonstrate that apple (Malus domestica) oligopeptide transporter3 (MdOPT3) mRNA can be transported over a long distance, from the leaf to the root, to regulate iron uptake; however, the mRNA of Arabidopsis (Arabidopsis thaliana) oligopeptide transporter 3 (AtOPT3), the MdOPT3 homolog from A. thaliana, does not move from shoot to root. Reciprocal heterologous expression of the two types of mRNAs showed that the immobile AtOPT3 became mobile and moved from the shoot to the root in two woody species, Malus and Populus, while the mobile MdOPT3 became immobile in two herbaceous species, A. thaliana and tomato (Solanum lycopersicum). Furthermore, we demonstrated that the different transmissibility of OPT3 in A. thaliana and Malus might be caused by divergence in RNA-binding proteins between herbaceous and woody plants. This study provides insights into mechanisms underlying differences in mRNA mobility and validates the important physiological functions associated with this process.

Combined analyses of translatome and transcriptome in Arabidopsis reveal new players responding to magnesium deficiency.

Translational control of gene expression, including recruitment of ribosomes to messenger RNA (mRNA), is particularly important during the response to stress. Purification of ribosome-associated mRNAs using translating ribosome affinity purification (TRAP) followed by RNA-sequencing facilitates the study of mRNAs undergoing active transcription and better proxies the translatome, or protein response, to stimuli. To identify plant responses to Magnesium (Mg) deficiency at the translational level, we combined transcriptome and translatome analyses. Excitingly, we found 26 previously unreported Mg-responsive genes that were only regulated at the translational level and not the transcriptional level, during the early response to Mg deficiency. In addition, mutants of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), the H+ /CATION EXCHANGER 1 and 3 (CAX1 and CAX3), and UBIQUITIN 11 (UBQ11) exhibited early chlorosis phenotype under Mg deficiency, supporting their functional involvement in ion homeostasis. Overall, our study strongly supports that TRAP-seq combined with RNA-seq followed by phenotype screening could facilitate the identification of novel players during stress responses.

Phloem unloading via the apoplastic pathway is essential for shoot distribution of root-synthesized cytokinins.

Root-synthesized cytokinins are transported to the shoot and regulate the growth, development, and stress responses of aerial tissues. Previous studies have demonstrated that Arabidopsis (Arabidopsis thaliana) ATP binding cassette (ABC) transporter G family member 14 (AtABCG14) participates in xylem loading of root-synthesized cytokinins. However, the mechanism by which these root-derived cytokinins are distributed in the shoot remains unclear. Here, we revealed that AtABCG14-mediated phloem unloading through the apoplastic pathway is required for the appropriate shoot distribution of root-synthesized cytokinins in Arabidopsis. Wild-type rootstocks grafted to atabcg14 scions successfully restored trans-zeatin xylem loading. However, only low levels of root-synthesized cytokinins and induced shoot signaling were rescued. Reciprocal grafting and tissue-specific genetic complementation demonstrated that AtABCG14 disruption in the shoot considerably increased the retention of root-synthesized cytokinins in the phloem and substantially impaired their distribution in the leaf apoplast. The translocation of root-synthesized cytokinins from the xylem to the phloem and the subsequent unloading from the phloem is required for the shoot distribution and long-distance shootward transport of root-synthesized cytokinins. This study revealed a mechanism by which the phloem regulates systemic signaling of xylem-mediated transport of root-synthesized cytokinins from the root to the shoot.

Unraveling the Roles of Vascular Proteins Using Proteomics.

Vascular bundles play important roles in transporting nutrients, growth signals, amino acids, and proteins between aerial and underground tissues. In order to understand these sophisticated processes, a comprehensive analysis of the roles of the components located in the vascular tissues is required. A great deal of data has been obtained from proteomic analyses of vascular tissues in plants, which mainly aim to identify the proteins moving through the vascular tissues. Here, different aspects of the phloem and xylem proteins are reviewed, including their collection methods, and their main biological roles in growth, and biotic and abiotic stress responses. The study of vascular proteomics shows great potential to contribute to our understanding of the biological mechanisms related to development and defense in plants.

Physio-morphological, biochemical, and anatomical traits of drought-tolerant and susceptible sorghum cultivars under pre- and post-anthesis drought.

Understanding the physiological mechanisms that control drought tolerance in crop plants is vital for effective breeding. In this study, we characterized drought stress responses in four sorghum cultivars exhibiting differential levels of drought tolerance at pre- and post-anthesis. Greenhouse-grown plants were subjected to two types of drought treatment, water stress (WS) and desiccant-induced water stress (DA), timed to occur at pre- and post-anthesis. Multiple physiological measurements were then made revealing varying responses among the experimental cultivars. The pre- and post-flowering drought-tolerant cultivar P898012 showed a significantly higher net photosynthetic rate, higher transpiration rate, and greater stomatal conductance compared to the drought-susceptible cultivars at both pre- and post-anthesis. A significantly greater stomatal size was also detected in P898012, while the highest stomatal density was found in the drought-susceptible cultivar P721Q. Meanwhile, the two post-flowering drought-tolerant cultivars P898082 and B35 had a higher starch content and exhibited greater osmotic potential under post-anthesis water stress. Compared to WS and well-watered control plants, a greater increase in root biomass was observed in P898012 under DA at pre-anthesis. This finding suggests that plants invested more assimilates into the roots under severe DA at pre-anthesis. Overall, our results show good conformity between drought tolerance in sorghum and key physiological mechanisms of stomatal conductance, root growth patterns, and starch accumulation, all of which act as coping mechanisms during critical drought-sensitive growth stages.

Paper-Based Analytical Devices for the Rapid and Direct Electrochemical Detection of Hydrogen Peroxide in Tomato Leaves Inoculated with Botrytis cinerea.

Hydrogen peroxide (H2O2) is an important signaling molecule and plays key roles in multiple plant physiological processes. The rapid and direct monitoring of H2O2 could improve our understanding of its regulatory mechanisms in plants. In this study, we developed a paper-based analytical device consisting of a disposable nano-gold modified indium tin oxide working electrode to provide a platform for the rapid and direct detection of H2O2. The total analytical time was dramatically shortened to be approximate 3 min due to the avoidance of the time-consuming and complex treatment of plant samples. In addition, the amount of plant samples required was less than 3 mg in our approach. We used this system to monitor the concentrations of H2O2 in tomato leaves infected by Botrytiscinerea within 24 h. Our results showed that the concentration of H2O2 in tomato leaves was increased in the initial phase, peaked at 1.5 µmol gFW-1 at 6 h, and then decreased. The production trend of H2O2 in tomato leaves inoculated with Botrytiscinerea detected with our approach is similar to the 3,3-diaminobenzidine staining method. Taken together, our study offers a rapid and direct approach for the detection of H2O2, which will not only pave the way for the further investigation of the regulation mechanisms of H2O2 in plants, but also promote the development of precision agriculture technology.

Long-Distance Movement of Mineral Deficiency-Responsive mRNAs in Nicotiana Benthamiana/Tomato Heterografts.

Deficiencies in essential mineral nutrients such as nitrogen (N), phosphorus (P), and iron (Fe) severely limit plant growth and crop yield. It has been discovered that both the local sensing system in roots and shoot-to-root systemic signaling via the phloem are involved in the regulation of the adaptive alterations in roots, in response to mineral deficiency. mRNAs are one group of molecules with systemic signaling functions in response to intrinsic and environmental cues; however, the importance of shoot-to-root mobile mRNAs stimulated by low mineral levels is not fully understood. In this study, we established a Nicotiana benthamiana/tomato heterograft system to identify shoot-to-root mobile mRNAs that are produced in response to low N, P or Fe. Multiple long-distance mobile mRNAs were identified to be associated with low mineral levels and a few of them may play important roles in hormonal metabolism and root architecture alteration. A comparison of the mobile mRNAs from our study with those identified from previous studies showed that very few transcripts are conserved among different species.

Long-Distance Movement of mRNAs in Plants.

Long-distance transport of information molecules in the vascular tissues could play an important role in regulating plant growth and enabling plants to cope with adverse environments. Various molecules, including hormones, proteins, small peptides and small RNAs have been detected in the vascular system and proved to have systemic signaling functions. Sporadic studies have shown that a number of mRNAs produced in the mature leaves leave their origin cells and move to distal tissues to exert important physiological functions. In the last 3-5 years, multiple heterograft systems have been developed to demonstrate that a large quantity of mRNAs are mobile in plants. Further comparison of the mobile mRNAs identified from these systems showed that the identities of these mRNAs are very diverse. Although species-specific mRNAs may regulate the unique physiological characteristic of the plant, mRNAs with conserved functions across multiple species are worth more effort in identifying universal physiological mechanisms existing in the plant kingdom.

WRKY18 and WRKY53 Coordinate with HISTONE ACETYLTRANSFERASE1 to Regulate Rapid Responses to Sugar.

Sugars provide a source of energy; they also function as signaling molecules that regulate gene expression, affect metabolism, and alter growth in plants. Rapid responses to sugar signaling and metabolism are essential for optimal growth and fitness, but the regulatory mechanisms underlying these are largely unknown. In this study, we found that the rapid induction of sugar responses in Arabidopsis (Arabidopsis thaliana) requires the W-box cis-elements in the promoter region of GLC 6-PHOSPHATE/PHOSPHATE TRANSLOCATOR2, a well-studied sugar response marker gene. The transcription factors WRKY18 and WRKY53 directly bind to the W-Box cis-elements in the promoter region of sugar response genes and activate their expression. In addition, HISTONE ACETYLTRANSFERASE 1 (HAC1) is recruited to the WRKY18 and WRKY53 complex that resides on the promoters. In this complex, HAC1 facilitates the acetylation of histone 3 Lys 27 (H3K27ac) on the sugar-responsive genes. Taken together, our findings demonstrate a mechanism by which sugar regulates chromatin modification and gene expression, thus helping plants to adjust their growth in response to environmental changes.

Identification of Phloem Mobile mRNAs Using the Solanaceae Heterograft System.

Large numbers of mRNAs move in the phloem and some may function as signals to exert important physiological functions in the distal recipient organs. Generating an authentic list of phloem mobile mRNA is a prerequisite for elucidating their physiological functions. Nicotiana benthamiana can be used as a scion to graft on a tomato (Solanum lycopersicum) rootstock. Thereby, shoot-to-root mobile N. benthamiana mRNAs transported via the phloem can be identified from the root of the tomato rootstock. Due to the close relationship and similar genome sequences of the two species, stringent informatics procedures should be applied to avoid false identification. This heterograft system can be used to study physiological processes associated with mRNAs that are mobile under either normal or adverse growth condition.

Physiological and Proteomic Responses of Mulberry Trees (Morus alba. L.) to Combined Salt and Drought Stress.

Intensive investigations have been conducted on the effect of sole drought or salinity stress on the growth of plants. However, there is relatively little knowledge on how plants, particularly woody species, respond to a combination of these two stresses although these stresses can simultaneously occur in the field. In this study, mulberry, an economically important resource for traditional medicine, and the sole food of domesticated silkworms was subjected to a combination of salt and drought stress and analyzed by physiological methods and TMT-based proteomics. Stressed mulberry exhibited significant alteration in physiological parameters, including root/shoot ratio, chlorophyll fluorescence, total carbon, and ion reallocation. A total of 577 and 270 differentially expressed proteins (DEPs) were identified from the stressed leaves and roots, respectively. Through KEGG analysis, these DEPs were assigned to multiple pathways, including carbon metabolism, photosynthesis, redox, secondary metabolism, and hormone metabolism. Among these pathways, the sucrose related metabolic pathway was distinctly enriched in both stressed leaves and roots, indicating an important contribution in mulberry under stress condition. The results provide a comprehensive understanding of the adaptive mechanism of mulberry in response to salt and drought stress, which will facilitate further studies on innovations in terms of crop performance.

Editing of the OsACS locus alters phosphate deficiency-induced adaptive responses in rice seedlings.

Phosphate (Pi) deficiency severely influences the growth and reproduction of plants. To cope with Pi deficiency, plants initiate morphological and biochemical adaptive responses upon sensing low Pi in the soil, and the plant hormone ethylene plays a crucial role during this process. However, how regulation of ethylene biosynthesis influences the Pi-induced adaptive responses remains unclear. Here, we determine the roles of rice 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), the rate-limiting enzymes in ethylene biosynthesis, in response to Pi deficiency. Through analysis of tissue-specific expression of OsACS in response to Pi deficiency and OsACS mutants generated by CRISPR/Cas9 [clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9] genome editing, we found that two members of the OsACS family, i.e. OsACS1 and OsACS2, are involved but differed in their importance in controlling the remodeling of root system architecture, transcriptional regulation of Pi starvation-induced genes, and cellular phosphorus homeostasis. Interestingly, in contrast to the known inhibitory role of ethylene on root elongation, both OsACS mutants, especially OsACS1, almost fail to promote lateral root growth in response to Pi deficiency, demonstrating a stimulatory role for ethylene in lateral root development under Pi-deficient conditions. Together, this study provides new insights into the roles of ethylene in Pi deficiency response in rice seedlings and the isoform-specific function of OsACS genes in this process.

Tissue-specific transcriptomic profiling of Plantago major provides insights for the involvement of vasculature in phosphate deficiency responses.

The vasculature of higher plants is important with transport of both nutrient and information molecules. To understand the correspondence of this tissue in molecular responses under phosphate (Pi) deficiency, Plantago major, a model plant for vasculature biology study, was chosen in our analysis. After RNA-Seq and de novo transcriptome assembly of 24 libraries prepared from the vasculature of P. major, 37,309 unigenes with a mean length of 1571 base pairs were obtained. Upon 24 h of Pi deficiency, 237 genes were shown to be differentially expressed in the vasculature of P. major. Among these genes, only 27 have been previously identified to be specifically expressed in the vasculature tissues in other plant species. Temporal expression of several marker genes associated with Pi deficiency showed that the time period of first 24 h is at the beginning stage of more dynamic expression patterns. In this study, we found several physiological processes, e.g., "phosphate metabolism and remobilization", "sucrose metabolism, loading and synthesis", "plant hormone metabolism and signal transduction", "transcription factors", and "metabolism of other minerals", were mainly involved in early responses to Pi deficiency in the vasculature. A number of vasculature genes with promising roles in Pi deficiency adaptation have been identified and deserve further functional characterization. This study clearly demonstrated that plant vasculature is actively involved in Pi deficiency responses and understanding of this process may help to create plants proficient to offset Pi deficiency.

Transcriptomic and functional analysis of cucumber (Cucumis sativus L.) fruit phloem during early development.

The phloem of the Cucurbitaceae has long been a subject of interest due to its complex nature and the economic importance of the family. As in a limited number of other families, cucurbit phloem is bicollateral, i.e. with sieve tubes on both sides of the xylem. To date little is known about the specialized functions of the internal phloem (IP) and external phloem (EP). Here, a combination of microscopy, fluorescent dye transport analysis, micro-computed tomography, laser capture microdissection and RNA-sequencing (RNA-Seq) were used to study the functions of IP and EP in the vascular bundles (VBs) of cucumber fruit. There is one type of VB in the peduncle, but four in the fruit: peripheral (PeVB), main (MVB), carpel (CVB) and placental (PlVB). The VBs are bicollateral, except for the CVB and PlVB. Phloem mobile tracers and 14 C applied to leaves are transported primarily in the EP, and to a lesser extent in the IP. RNA-Seq data indicate preferential gene transcription in the IP related to differentiation/development, hormone transport, RNA or protein modification/processing/transport, and nitrogen compound metabolism and transport. The EP preferentially expresses genes for stimulus/stress, defense, ion transport and secondary metabolite biosynthesis. The MVB phloem is preferentially involved in photoassimilate transport, unloading and long-distance signaling, while the PeVB plays a more substantial role in morphogenesis and/or development and defense response. CVB and PlVB transcripts are biased toward development of reproductive organs. These findings provide an integrated view of the differentiated structure and function of the vascular tissue in cucumber fruit.

Physiological and transcriptomic responses of reproductive stage soybean to drought stress.

KEY MESSAGE: The dynamic alterations of the physiological and molecular processes in reproductive stage soybean indicated the dramatic impact caused by drought. Drought is a major abiotic stress that limits soybean (Glycine max) production. Most prior studies were focused on either model species or crops that are at their vegetative stages. It is known that the reproductive stage of soybean is more susceptible to drought. Therefore, an understanding on the responsive mechanisms during this stage will not only be important for basic plant physiology, but the knowledge can also be used for crop improvement via either genetic engineering or molecular breeding. In this study, physiological measurements and RNA-Seq analysis were used to dissect the metabolic alterations and molecular responses in the leaves of soybean grown at drought condition. Photosynthesis rate, stomata conductance, transpiration, and water potential were reduced. The activities of SOD and CAT were increased, while the activity of POD stayed unchanged. A total of 2771 annotated genes with at least twofold changes were found to be differentially expressed in the drought-stressed plants in which 1798 genes were upregulated and 973 were downregulated. Via KEGG analysis, these genes were assigned to multiple molecular pathways, including ABA biogenesis, compatible compound accumulation, secondary metabolite synthesis, fatty acid desaturation, plant transcription factors, etc. The large number of differentially expressed genes and the diverse pathways indicated that soybean employs complicated mechanisms to cope with drought. Some of the identified genes and pathways can be used as targets for genetic engineering or molecular breeding to improve drought resistance in soybean.

Elucidation of the Mechanisms of Long-Distance mRNA Movement in a Nicotiana benthamiana/Tomato Heterograft System.

Recent heterograft analyses showed that large-scale messenger RNA (mRNA) movement takes place in the phloem, but the number of mobile transcripts reported varies widely. However, our knowledge of the mechanisms underlying large-scale mRNA movement remains limited. In this study, using a Nicotiana benthamiana/tomato (Solanum lycopersicum) heterograft system and a transgenic approach involving potato (Solanum tuberosum), we found that: (1) the overall mRNA abundance in the leaf is not a good indicator of transcript mobility to the root; (2) increasing the expression levels of nonmobile mRNAs in the companion cells does not promote their mobility; (3) mobile mRNAs undergo degradation during their movement; and (4) some mRNAs arriving in roots move back to shoots. These results indicate that mRNA movement has both regulated and unregulated components. The cellular origins of mobile mRNAs may differ between herbaceous and woody species. Taken together, these findings suggest that the long-distance movement of mRNAs is a complex process and that elucidating the physiological roles associated with this movement is challenging but remains an important task for future research.