Plant 22-nt siRNAs mediate translational repression and stress adaptation.

Small interfering RNAs (siRNAs) are essential for proper development and immunity in eukaryotes1. Plants produce siRNAs with lengths of 21, 22 or 24 nucleotides. The 21- and 24-nucleotide species mediate cleavage of messenger RNAs and DNA methylation2,3, respectively, but the biological functions of the 22-nucleotide siRNAs remain unknown. Here we report the identification and characterization of a group of endogenous 22-nucleotide siRNAs that are generated by the DICER-LIKE 2 (DCL2) protein in plants. When cytoplasmic RNA decay and DCL4 are deficient, the resulting massive accumulation of 22-nucleotide siRNAs causes pleiotropic growth disorders, including severe dwarfism, meristem defects and pigmentation. Notably, two genes that encode nitrate reductases-NIA1 and NIA2-produce nearly half of the 22-nucleotide siRNAs. Production of 22-nucleotide siRNAs triggers the amplification of gene silencing and induces translational repression both gene specifically and globally. Moreover, these 22-nucleotide siRNAs preferentially accumulate upon environmental stress, especially those siRNAs derived from NIA1/2, which act to restrain translation, inhibit plant growth and enhance stress responses. Thus, our research uncovers the unique properties of 22-nucleotide siRNAs, and reveals their importance in plant adaptation to environmental stresses.

A comprehensive workflow for optimizing RNA-seq data analysis.

BACKGROUND: Current RNA-seq analysis software for RNA-seq data tends to use similar parameters across different species without considering species-specific differences. However, the suitability and accuracy of these tools may vary when analyzing data from different species, such as humans, animals, plants, fungi, and bacteria. For most laboratory researchers lacking a background in information science, determining how to construct an analysis workflow that meets their specific needs from the array of complex analytical tools available poses a significant challenge. RESULTS: By utilizing RNA-seq data from plants, animals, and fungi, it was observed that different analytical tools demonstrate some variations in performance when applied to different species. A comprehensive experiment was conducted specifically for analyzing plant pathogenic fungal data, focusing on differential gene analysis as the ultimate goal. In this study, 288 pipelines using different tools were applied to analyze five fungal RNA-seq datasets, and the performance of their results was evaluated based on simulation. This led to the establishment of a relatively universal and superior fungal RNA-seq analysis pipeline that can serve as a reference, and certain standards for selecting analysis tools were derived for reference. Additionally, we compared various tools for alternative splicing analysis. The results based on simulated data indicated that rMATS remained the optimal choice, although consideration could be given to supplementing with tools such as SpliceWiz. CONCLUSION: The experimental results demonstrate that, in comparison to the default software parameter configurations, the analysis combination results after tuning can provide more accurate biological insights. It is beneficial to carefully select suitable analysis software based on the data, rather than indiscriminately choosing tools, in order to achieve high-quality analysis results more efficiently.

PTI-ETI synergistic signal mechanisms in plant immunity.

Plants face a relentless onslaught from a diverse array of pathogens in their natural environment, to which they have evolved a myriad of strategies that unfold across various temporal scales. Cell surface pattern recognition receptors (PRRs) detect conserved elicitors from pathogens or endogenous molecules released during pathogen invasion, initiating the first line of defence in plants, known as pattern-triggered immunity (PTI), which imparts a baseline level of disease resistance. Inside host cells, pathogen effectors are sensed by the nucleotide-binding/leucine-rich repeat (NLR) receptors, which then activate the second line of defence: effector-triggered immunity (ETI), offering a more potent and enduring defence mechanism. Moreover, PTI and ETI collaborate synergistically to bolster disease resistance and collectively trigger a cascade of downstream defence responses. This article provides a comprehensive review of plant defence responses, offering an overview of the stepwise activation of plant immunity and the interactions between PTI-ETI synergistic signal transduction.

The miR6445-NAC029 module regulates drought tolerance by regulating the expression of glutathione S-transferase U23 and reactive oxygen species scavenging in Populus.

MicroRNAs are essential in plant development and stress resistance, but their specific roles in drought stress require further investigation. Here, we have uncovered that a Populus-specific microRNAs (miRNA), miR6445, targeting NAC (NAM, ATAF, and CUC) family genes, is involved in regulating drought tolerance of poplar. The expression level of miR6445 was significantly upregulated under drought stress; concomitantly, seven targeted NAC genes showed significant downregulation. Silencing the expression of miR6445 by short tandem target mimic technology significantly decreased the drought tolerance in poplar. Furthermore, 5' RACE experiments confirmed that miR6445 directly targeted NAC029. The overexpression lines of PtrNAC029 (OE-NAC029) showed increased sensitivity to drought compared with knockout lines (Crispr-NAC029), consistent with the drought-sensitive phenotype observed in miR6445-silenced strains. PtrNAC029 was further verified to directly bind to the promoters of glutathione S-transferase U23 (GSTU23) and inhibit its expression. Both Crispr-NAC029 and PtrGSTU23 overexpressing plants showed higher levels of PtrGSTU23 transcript and GST activity while accumulating less reactive oxygen species (ROS). Moreover, poplars overexpressing GSTU23 demonstrated enhanced drought tolerance. Taken together, our research reveals the crucial role of the miR6445-NAC029-GSTU23 module in enhancing poplar drought tolerance by regulating ROS homeostasis. This finding provides new molecular targets for improving the drought resistance of trees.

An alternative splicing variant of PtRD26 delays leaf senescence by regulating multiple NAC transcription factors in Populus.

During leaf senescence, the final stage of leaf development, nutrients are recycled from leaves to other organs, and therefore proper control of senescence is thus critical for plant fitness. Although substantial progress has been achieved in understanding leaf senescence in annual plants, the molecular factors that control leaf senescence in perennial woody plants are largely unknown. Using RNA sequencing, we obtained a high-resolution temporal profile of gene expression during autumn leaf senescence in poplar (Populus tomentosa). Identification of hub transcription factors (TFs) by co-expression network analysis of genes revealed that senescence-associated NAC family TFs (Sen-NAC TFs) regulate autumn leaf senescence. Age-dependent alternative splicing (AS) caused an intron retention (IR) event in the pre-mRNA encoding PtRD26, a NAC-TF. This produced a truncated protein PtRD26IR, which functions as a dominant-negative regulator of senescence by interacting with multiple hub Sen-NAC TFs, thereby repressing their DNA-binding activities. Functional analysis of senescence-associated splicing factors identified two U2 auxiliary factors that are involved in AS of PtRD26IR. Correspondingly, silencing of these factors decreased PtRD26IR transcript abundance and induced early senescence. We propose that an age-dependent increase of IR splice variants derived from Sen-NAC TFs is a regulatory program to fine tune the molecular mechanisms that regulate leaf senescence in trees.

Aspartyl tRNA-synthetase (AspRS) gene family enhances drought tolerance in poplar through BABA-PtrIBIs-PtrVOZ signaling module.

BACKGROUND: Drought stress is a prevalent abiotic stress that significantly hinders the growth and development of plants. According to studies, ß-aminobutyric acid (BABA) can influence the ABA pathway through the AtIBI1 receptor gene to enhance cold resistance in Arabidopsis. However, the Aspartate tRNA-synthetase (AspRS) gene family, which acts as the receptor for BABA, has not yet been investigated in poplar. Particularly, it is uncertain how the AspRS gene family (PtrIBIs)r can resist drought stress after administering various concentrations of BABA to poplar. RESULTS: In this study, we have identified 12 AspRS family genes and noted that poplar acquired four PtrIBI pairs through whole genome duplication (WGD). We conducted cis-action element analysis and found a significant number of stress-related action elements on different PtrIBI genes promoters. The expression of most PtrIBI genes was up-regulated under beetle and mechanical damage stresses, indicating their potential role in responding to leaf damage stress. Our results suggest that a 50 mM BABA treatment can alleviate the damage caused by drought stress in plants. Additionally, via transcriptome sequencing, we observed that the partial up-regulation of BABA receptor genes, PtrIBI2/4/6/8/11, in poplars after drought treatment. We hypothesize that poplar responds to drought stress through the BABA-PtrIBIs-PtrVOZ coordinated ABA signaling pathway. Our research provides molecular evidence for understanding how plants respond to drought stress through external application of BABA. CONCLUSIONS: In summary, our study conducted genome-wide analysis of the AspRS family of P. trichocarpa and identified 12 PtrIBI genes. We utilized genomics and bioinformatics to determine various characteristics of PtrIBIs such as chromosomal localization, evolutionary tree, gene structure, gene doubling, promoter cis-elements, and expression profiles. Our study found that certain PtrIBI genes are regulated by drought, beetle, and mechanical damage implying their crucial role in enhancing poplar stress tolerance. Additionally, we observed that external application of low concentrations of BABA increased plant drought resistance under drought stress. Through the BABA-PtrIBIs-PtrVOZ signaling module, poplar plants were able to transduce ABA signaling and regulate their response to drought stress. These results suggest that the PtrIBI genes in poplar have the potential to improve drought tolerance in plants through the topical application of low concentrations of BABA.

The Protective Role of Non-Photochemical Quenching in PSII Photo-Susceptibility: A Case Study in the Field.

Non-photochemical quenching (NPQ) has been regarded as a safety valve to dissipate excess absorbed light energy not used for photochemistry. However, there exists no general consensus on the photoprotective role of NPQ. In the present study, we quantified the Photosystem II (PSII) photo-susceptibilities (mpi) in the presence of lincomycin, under red light given to five shade-acclimated tree species grown in the field. Photosynthetic energy partitioning theory was applied to investigate the relationships between mpi and each of the regulatory light-induced NPQ [Y(NPQ)], the quantum yield of the constitutive nonregulatory NPQ [Y(NO)] and the PSII photochemical yield in the light-adapted state [Y(PSII)] under different red irradiances. It was found that in the low to moderate irradiance range (50-800 µmol m-2 s-1) when the fraction of open reaction centers (qP) exceeded 0.4, mpi exhibited no association with Y(NPQ), Y(NO) and Y(PSII) across species. However, when qP < 0.4 (1,500 µmol m-2 s-1), there existed positive relationships between mpi and Y(NPQ) or Y(NO) but a negative relationship between mpi and Y(PSII). It is postulated that both Y(NPQ) and Y(NO) contain protective and damage components and that using only Y(NPQ) or Y(NO) metrics to identify the photo-susceptibility of a species is a risk. It seems that qP regulates the balance of the two components for each of Y(NPQ) and Y(NO). Under strong irradiance, when both protective Y(NPQ) and Y(NO) are saturated/depressed, the forward electron flow [i.e. Y(PSII)] acts as the last defense to resist photoinhibition.

Geographical variation in functional traits of leaves of Caryopteris mongholica and the role of climate.

BACKGROUND: Quantifying intra-specific variation in leaf functional traits along environmental gradients is important for understanding species' responses to climate change. In this study, we assessed the degree of among and within populations variation in leaf functional traits and explored leaf response to geographic and climate change using Caryopteris mongholica as material, which has a wide range of distribution environments. RESULTS: We selected 40 natural populations of C. mongholica, measured 8 leaf functional traits, analyzed the extent of trait variation among and within populations, and developed geographic and climatic models to explain trait variation between populations. Our results showed that the variation in leaf functional traits of C. mongholica was primarily lower within populations compared to among populations. Specifically, the leaf area (LA) exhibited higher variability both among and within populations, whereas leaf carbon content (LC) exhibited lower variation within populations but greater variation among populations. We observed a specific covariation pattern among traits and a strong linkage between morphological, economic, and mechanical traits. Increasing minimum temperature, precipitation of month, and seasonal precipitation differences all limited the growth and development of C. mongholica. However, it was observed that an increase in mean annual precipitation positively influenced the morphological development of its leaf. CONCLUSIONS: These results demonstrate the response of intra-specific trait variation to the environment and provide valuable insights into the adaptation of intra-specific leaf functional traits under changing climatic conditions.

Identification of WUSCHEL-related homeobox gene and truncated small peptides in transformation efficiency improvement in Eucalyptus.

BACKGROUND: The WUSCHEL-related Homeobox (WOX) genes, which encode plant-specific homeobox (HB) transcription factors, play crucial roles in regulating plant growth and development. However, the functions of WOX genes are little known in Eucalyptus, one of the fastest-growing tree resources with considerable widespread cultivation worldwide. RESULTS: A total of nine WOX genes named EgWOX1-EgWOX9 were retrieved and designated from Eucalyptus grandis. From the three divided clades marked as Modern/WUS, Intermediate and Ancient, the largest group Modern/WUS (6 EgWOXs) contains a specific domain with 8 amino acids: TLQLFPLR. The collinearity, cis-regulatory elements, protein-protein interaction network and gene expression analysis reveal that the WUS proteins in E. grandis involve in regulating meristems development and regeneration. Furthermore, by externally adding of truncated peptides isolated from WUS specific domain, the transformation efficiency in E. urophylla × E. grandis DH32-29 was significant enhanced. The transcriptomics data further reveals that the use of small peptides activates metabolism pathways such as starch and sucrose metabolism, phenylpropanoid biosynthesis and flavonoid biosynthesis. CONCLUSIONS: Peptides isolated from WUS protein can be utilized to enhance the transformation efficiency in Eucalyptus, thereby contributing to the high-efficiency breeding of Eucalyptus.

Spliceosomal protein U2B″ delays leaf senescence by enhancing splicing variant JAZ9ß expression to attenuate jasmonate signaling in Arabidopsis.

The regulatory framework of leaf senescence is gradually becoming clearer; however, the fine regulation of this process remains largely unknown. Here, genetic analysis revealed that U2 small nuclear ribonucleoprotein B (U2B″), a component of the spliceosome, is a negative regulator of leaf senescence. Mutation of U2B″ led to precocious leaf senescence, whereas overexpression of U2B″ extended leaf longevity. Transcriptome analysis revealed that the jasmonic acid (JA) signaling pathway was activated in the u2b″ mutant. U2B″ enhances the generation of splicing variant JASMONATE ZIM-DOMAIN 9ß (JAZ9ß) with an intron retention in the Jas motif, which compromises its interaction with CORONATINE INSENSITIVE1 and thus enhances the stability of JAZ9ß protein. Moreover, JAZ9ß could interact with MYC2 and obstruct its activity, thereby attenuating JA signaling. Correspondingly, overexpression of JAZ9ß rescued the early senescence phenotype of the u2b″ mutant. Furthermore, JA treatment promoted expression of U2B″ that was found to be a direct target of MYC2. Overexpression of MYC2 in the u2b″ mutant resulted in a more pronounced premature senescence than that in wild-type plants. Collectively, our findings reveal that the spliceosomal protein U2B″ fine-tunes leaf senescence by enhancing the expression of JAZ9ß and thereby attenuating JA signaling.

Histone variant HTB4 delays leaf senescence by epigenetic control of Ib bHLH transcription factor-mediated iron homeostasis.

Leaf senescence is an orderly process regulated by multiple internal factors and diverse environmental stresses including nutrient deficiency. Histone variants are involved in regulating plant growth and development. However, their functions and underlying regulatory mechanisms in leaf senescence remain largely unclear. Here, we found that H2B histone variant HTB4 functions as a negative regulator of leaf senescence. Loss of function of HTB4 led to early leaf senescence phenotypes that were rescued by functional complementation. RNA-seq analysis revealed that several Ib subgroup basic helix-loop-helix (bHLH) transcription factors (TFs) involved in iron (Fe) homeostasis, including bHLH038, bHLH039, bHLH100, and bHLH101, were suppressed in the htb4 mutant, thereby compromising the expressions of FERRIC REDUCTION OXIDASE 2 (FRO2) and IRON-REGULATED TRANSPORTER (IRT1), two important components of the Fe uptake machinery. Chromatin immunoprecipitation-quantitative polymerase chain reaction analysis revealed that HTB4 could bind to the promoter regions of Ib bHLH TFs and enhance their expression by promoting the enrichment of the active mark H3K4me3 near their transcriptional start sites. Moreover, overexpression of Ib bHLH TFs or IRT1 suppressed the premature senescence phenotype of the htb4 mutant. Our work established a signaling pathway, HTB4-bHLH TFs-FRO2/IRT1-Fe homeostasis, which regulates the onset and progression of leaf senescence.

Crucial Abiotic Stress Regulatory Network of NF-Y Transcription Factor in Plants.

Nuclear Factor-Y (NF-Y), composed of three subunits NF-YA, NF-YB and NF-YC, exists in most of the eukaryotes and is relatively conservative in evolution. As compared to animals and fungi, the number of NF-Y subunits has significantly expanded in higher plants. The NF-Y complex regulates the expression of target genes by directly binding the promoter CCAAT box or by physical interaction and mediating the binding of a transcriptional activator or inhibitor. NF-Y plays an important role at various stages of plant growth and development, especially in response to stress, which attracted many researchers to explore. Herein, we have reviewed the structural characteristics and mechanism of function of NF-Y subunits, summarized the latest research on NF-Y involved in the response to abiotic stresses, including drought, salt, nutrient and temperature, and elaborated the critical role of NF-Y in these different abiotic stresses. Based on the summary above, we have prospected the potential research on NF-Y in response to plant abiotic stresses and discussed the difficulties that may be faced in order to provide a reference for the in-depth analysis of the function of NF-Y transcription factors and an in-depth study of plant responses to abiotic stress.

Genome-Wide Analysis of the FBA Subfamily of the Poplar F-Box Gene Family and Its Role under Drought Stress.

F-box proteins are important components of eukaryotic SCF E3 ubiquitin ligase complexes, which specifically determine protein substrate proteasomal degradation during plant growth and development, as well as biotic and abiotic stress. It has been found that the FBA (F-box associated) protein family is one of the largest subgroups of the widely prevalent F-box family and plays significant roles in plant development and stress response. However, the FBA gene family in poplar has not been systematically studied to date. In this study, a total of 337 F-box candidate genes were discovered based on the fourth-generation genome resequencing of P. trichocarpa. The domain analysis and classification of candidate genes revealed that 74 of these candidate genes belong to the FBA protein family. The poplar F-box genes have undergone multiple gene replication events, particularly in the FBA subfamily, and their evolution can be attributed to genome-wide duplication (WGD) and tandem duplication (TD). In addition, we investigated the P. trichocarpa FBA subfamily using the PlantGenIE database and quantitative real-time PCR (qRT-PCR); the results showed that they are expressed in the cambium, phloem and mature tissues, but rarely expressed in young leaves and flowers. Moreover, they are also widely involved in the drought stress response. At last, we selected and cloned PtrFBA60 for physiological function analysis and found that it played an important role in coping with drought stress. Taken together, the family analysis of FBA genes in P. trichocarpa provides a new opportunity for the identification of P. trichocarpa candidate FBA genes and elucidation of their functions in growth, development and stress response, thus demonstrating their utility in the improvement of P. trichocarpa.

Genetic diversity and population structure of Caryopteris mongholica revealed by reduced representation sequencing.

BACKGROUND: Caryopteris mongholica Bunge is a rare broad-leaved shrub distributed in the desert and arid regions of Mongol and North China. Due to land reclamation, natural habitat deterioration and anthropogenic activities in recent years, the wild resources have sharply reduced. To effectively protect and rationally use it, we investigated the genetic diversity and population structure from 18 populations across the range of C. mongholica in China by reduced representation sequencing technology. RESULTS: We found the overall average values of observed heterozygosity (Ho), expected heterozygosity (He), and average nucleotide diversity (π) were 0.43, 0.35 and 0.135, respectively. Furthermore, the NM17 population exhibited higher genetic diversity than other populations. The phylogenetic tree, principal component analysis (PCA) and structure analysis showed the sampled individuals clustered into two main groups. The NM03 population, with individuals clustered in both groups, may be a transitional population located between the two groups. In addition, most genetic variation existed within populations (90.97%) compared to that among the populations (9.03%). Interestingly, geographic and environmental distances were almost equally important to the observed genetic differences. Redundancy analysis (RDA) identified optical radiation (OR), minimum temperature (MIT) and mean annual precipitation (MAP) related variables as the most important environment factors influencing genetic variation, and the importance of MIT was also confirmed in the latent factor mixed models (LFMM). CONCLUSIONS: The results of this study facilitate research on the genetic diversity of C. mongholica. These genetic features provided vital information for conserving and sustainably developing the C. mongholica genetic resources.

CLE42 delays leaf senescence by antagonizing ethylene pathway in Arabidopsis.

Leaf senescence is the final stage of leaf development and is influenced by numerous internal and environmental factors. CLE family peptides are plant-specific peptide hormones that regulate various developmental processes. However, the role of CLE in regulating Arabidopsis leaf senescence remains unclear. Here, we found that CLE42 is a negative regulator of leaf senescence by using a CRISPR/Cas9-produced CLE mutant collection. The cle42 mutant displayed earlier senescence phenotypes, while overexpression of CLE42 delayed age-dependent and dark-induced leaf senescence. Moreover, application of the synthesized 12-amino-acid peptide (CLE42p) also delayed leaf senescence under natural and dark conditions. CLE42 and CLE41/44 displayed functional redundancy in leaf senescence, and the cle41 cle42 cle44 triple mutant displayed more pronounced earlier senescence phenotypes than any single mutant. Analysis of differentially expressed genes obtained by RNA-Seq methodology revealed that the ethylene pathway was suppressed by overexpressing CLE42. Moreover, CLE42 suppressed ethylene biosynthesis and thus promoted the protein accumulation of EBF, which in turn decreased the function of EIN3. Accordingly, mutation of EIN3/EIL1 or overexpression of EBF1 suppressed the earlier senescence phenotypes of the cle42 mutant. Together, our results reveal that the CLE peptide hormone regulates leaf senescence by communicating with the ethylene pathway.

The transcription factor GNC optimizes nitrogen use efficiency and growth by up-regulating the expression of nitrate uptake and assimilation genes in poplar.

Plants have evolved complex mechanisms to cope with the fluctuating environmental availability of nitrogen. However, potential genes modulating plant responses to nitrate are yet to be characterized. Here, a poplar GATA transcription factor gene PdGNC (GATA nitrate-inducible carbon-metabolism-involved) was found to be strongly induced by low nitrate. Overexpressing PdGNC in poplar clone 717-1B4 (P. tremula × alba) significantly improved nitrate uptake, remobilization, and assimilation with higher nitrogen use efficiency (NUE) and faster growth, particularly under low nitrate conditions. Conversely, CRISPR/Cas9-mediated poplar mutant gnc exhibited decreased nitrate uptake, relocation, and assimilation, combined with lower NUE and slower growth. Assays with yeast one-hybrid, electrophoretic mobility shift, and a dual-luciferase reporter showed that PdGNC directly activated the promoters of nitrogen pathway genes PdNRT2.4b, PdNR, PdNiR, and PdGS2, leading to a significant increase in nitrate utilization in poplar. As expected, the enhanced NUE promoted growth under low nitrate availability. Taken together, our data show that PdGNC plays an important role in the regulation of NUE and growth in poplar by improving nitrate acquisition, remobilization, and assimilation, and provide a promising strategy for molecular breeding to improve productivity under nitrogen limitation in trees.

LSD 3.0: a comprehensive resource for the leaf senescence research community.

The leaf senescence database (LSD) is a comprehensive resource of senescence-associated genes (SAGs) and their corresponding mutants. Through manual curation and extensive annotation, we updated the LSD to a new version LSD 3.0, which contains 5853 genes and 617 mutants from 68 species. To provide sustainable and reliable services for the plant research community, LSD 3.0 (https://bigd.big.ac.cn/lsd/) has been moved to and maintained by the National Genomics Data Center at Beijing Institute of Genomics, Chinese Academy of Sciences. In the current release, we added some new features: (i) Transcriptome data of leaf senescence in poplar were integrated; (ii) Leaf senescence-associated transcriptome data information in Arabidopsis, rice and soybean were included; (iii) Senescence-differentially expressed small RNAs (Sen-smRNA) in Arabidopsis were identified; (iv) Interaction pairs between Sen-smRNAs and senescence-associated transcription factors (Sen-TF) were established; (v) Senescence phenotypes of 90 natural accessions (ecotypes) and 42 images of ecotypes in Arabidopsis were incorporated; (vi) Mutant seed information of SAGs in rice obtained from Kitbase was integrated; (vii) New options of search engines for ecotypes and transcriptome data were implemented. Together, the updated database bears great utility to continue to provide users with useful resources for studies of leaf senescence.

Advances in 5-Aminolevulinic Acid Priming to Enhance Plant Tolerance to Abiotic Stress.

Priming is an adaptive strategy that improves plant defenses against biotic and abiotic stresses. Stimuli from chemicals, abiotic cues, and pathogens can trigger the establishment of priming state. Priming with 5-aminolevulinic acid (ALA), a potential plant growth regulator, can enhance plant tolerance to the subsequent abiotic stresses, including salinity, drought, heat, cold, and UV-B. However, the molecular mechanisms underlying the remarkable effects of ALA priming on plant physiology remain to be elucidated. Here, we summarize recent progress made in the stress tolerance conferred by ALA priming in plants and provide the underlying molecular and physiology mechanisms of this phenomenon. Priming with ALA results in changes at the physiological, transcriptional, metabolic, and epigenetic levels, and enhances photosynthesis and antioxidant capacity, as well as nitrogen assimilation, which in turn increases the resistance of abiotic stresses. However, the signaling pathway of ALA, including receptors as well as key components, is currently unknown, which hinders the deeper understanding of the defense priming caused by ALA. In the future, there is an urgent need to reveal the molecular mechanisms by which ALA regulates plant development and enhances plant defense with the help of forward genetics, multi-omics technologies, as well as genome editing technology.

The Regulation of Nitrate Reductases in Response to Abiotic Stress in Arabidopsis.

The two homologous genes, NIA1 and NIA2, encode nitrate reductases in Arabidopsis, which govern the reduction of nitrate to nitrite. This step is the rate-limiting step of the nitrate assimilation and utilization. Therefore, the regulation of NIA1 and NIA2 is important for plant development and growth. Although they are similar in sequence and structure, their regulations are different. Genetic analysis uncovers that NIA1, rather than NIA2, plays a predominant role in adopting to ABA stress. Although both long-term stress conditions can cause an improvement in NIA1 levels, a decrease in NIA1 levels under short-term treatments seems to be necessary for plants to switch from the growth status into the adopting status. Interestingly, the downregulation of the NR is distinct under different stress conditions. Under ABA treatment, the NR proteins are degraded via a 26S-proteasome dependent manner, while the transcriptional regulation is the main manner to rapidly reduce the NIA1 levels under nitrogen deficiency and NaCl stress conditions. These results indicate that under stress conditions, the regulation of NIA1 is complex, and it plays a key role in regulating the balance between growth and adaptation.

Regulation of cytokinin biosynthesis using PtRD26pro -IPT module improves drought tolerance through PtARR10-PtYUC4/5-mediated reactive oxygen species removal in Populus.

Drought is a critical environmental factor which constrains plant survival and growth. Genetic engineering provides a credible strategy to improve drought tolerance of plants. Here, we generated transgenic poplar lines expressing the isopentenyl transferase gene (IPT) under the driver of PtRD26 promoter (PtRD26pro -IPT). PtRD26 is a senescence and drought-inducible NAC transcription factor. PtRD26pro -IPT plants displayed multiple phenotypes, including improved growth and drought tolerance. Transcriptome analysis revealed that auxin biosynthesis pathway was activated in the PtRD26pro -IPT plants, leading to an increase in auxin contents. Biochemical analysis revealed that ARABIDOPSIS RESPONSE REGULATOR10 (PtARR10), one of the type-B ARR transcription factors in the cytokinin pathway, was induced in PtRD26pro -IPT plants and directly regulated the transcripts of YUCCA4 (PtYUC4) and YUCCA5 (PtYUC5), two enzymes in the auxin biosynthesis pathway. Overexpression of PtYUC4 enhanced drought tolerance, while simultaneous silencing of PtYUC4/5 evidently attenuated the drought tolerance of PtRD26pro -IPT plants. Intriguingly, PtYUC4/5 displayed a conserved thioredoxin reductase activity that is required for drought tolerance by deterring reactive oxygen species accumulation. Our work reveals the molecular basis of cytokinin and auxin interactions in response to environmental stresses, and shed light on the improvement of drought tolerance without a growth penalty in trees by molecular breeding.