Molecular Plant

Overuse of fertilizers increased greenhouse gases emissions, induced global climate changes and extreme weather and made future agriculture unsustainable. Engineering crops to adapt to stressed conditions is crucial. Here, we cloned a transcription factor TabZIP45 (basic region zipper), controlled by a microRNA binding site polymorphism, conferring adaptation to both nitrogen deficiency and dense planting. TabZIP45 interacted with TaFTL43 (Flowering locus T like43) to change gene expression regulation. TabZIP45 coordinated phosphatidylinositol diphosphate (PIP2) metabolism and calcium (Ca2+) signaling to adapt to environmental stresses. Knockout of TabZIP45-4B by genome editing rescued grain yield loss caused by nitrogen deficiency by modulation of TaDwarf4 under dense planting through Ca2+ signaling disruption. Thus, TabZIP45-4B edited wheat warranted a sustainable and environmentally friendly way to enhance grain yield under adverse conditions. One-Sentence SummaryCalcium and lipids integrated adverse environmental signaling to modulate plant growth

Withdrawal StatementThe author has withdrawn this manuscript owing to it having been posted without securing the appropriate approvals. Therefore, the author does not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

Dear Editor, Since the first plant receptor-like kinase (RLK) gene ZmPK1 was cloned from Zea mays in 1990 (Walker & Zhang, 1990), this large gene family has been extensively studied and shown to play crucial roles in growth, development, and immunity (Tang et al., 2017). RLKs are widespread in the plant kingdom, while the biological functions of most RLKs remain largely elusive (Dievart et al., 2020). Given RLKs share a conserved monophyletic RLK/Pelle kinase domain, RLKs in several model plants are classified into distinct families by extracellular domains (ECDs) (Shiu & Bleecker, 2001). However, independent domain shuffling in specific lineages drives the origin of novel families, which raises a question: how about the landscape of RLKs in the whole plant kingdom? Previously, sequence homology-based methods have been widely used for RLK identification and classification, which probably will miss the distantly related proteins but with similar structures and potential novel families unmentioned in the literature. The academic community urgently requires a dedicated database for a systematic overview of the RLK gene family, providing data support for in-depth research on RLK genes. Here, we used a topology-based method to accurately isolate the RLKomes from proteomes. The obtained RLKomes were further classified into (sub)families based on ECD domains. We constructed a comprehensively curated plant RLK database (https://biotec.njau.edu.cn/rlkdb/), which contains valuable resources for investigating the origin and evolution of the RLK family and multiple online tools for personalized analysis.

Nutrients are important for growth in both plants and animals, uncovering of signaling pathway in nutrients determined growth is essential. Here we cloned TaPCGR1-3B (Phospholipid Coordinated Growth and nutrients Response 1), controlled by SNPs on alternative splicing and transcription factors binding, conferring nitrogen deficiency response. TaPCGR1-3B was localized in plasma membrane and endoplasmic reticulum of meristem cell. Nitrogen deprivation stimulated interaction of TaPCGR1-3B with G protein alpha subunit and phospholipase C 9, which was inhibited by phosphatidylcholine, to trigger Ca2+ signaling and inhibit normal growth. Knockdown of TaPCGR1 rescued the growth inhibition caused by nutrient deficient conditions by modulation of phosphatidylcholine induced growth gene expression through Ca2+ signaling disruption. Modulating of phosphatidylcholine mediated TaPCGR1 activity thus tightly regulated growth through Ca2+ signaling.

The protein concentration gradients of the master regulators of the root meristem, named the PLETHORA proteins, modulate the root meristem size. Root meristem growth factor 1 (RGF1) peptide extends the PLETHORA2 (PLT2) protein gradients by altering reactive oxygen species (ROS) distributions. However, the underlying mechanism through which the ROS alterations regulate PLT2 remains unknown. Here, we demonstrate that the 212th cysteine of the PLT2 protein plays a pivotal role in modulating PLT2 stability through the ROS altered by RGF1. The substitution of the 212th cysteine of PLT2 with serine (PLT2C212S) enhanced the PLT2 protein stability upon RGF1 and resulted in robust resistance to ROS relative to the native PLT2. Accordingly, PLT2C212S modulated expressions of certain specific root development-related genes to a greater extent than native PLT2. In summary, these findings show that the PLT2 concentration gradient formation through ROS, modulated by RGF1, is dependent on a mechanism involving the 212th cysteine of PLT2.

Remodeling of the actin cytoskeleton is a critical process for plant immunity, essential for the transport, activation, and stabilization of immune-regulatory molecules and organelles. In this process, actin depolymerization factors (ADFs) function as key players through severing and depolymerizing actin microfilaments. However, recent evidence suggests that ADFs may possess non-canonical immune functions inside the nucleus, in addition to the canonic cytosolic role, a phenomenon not adequately explained by the traditional mechanistic model of ADF-actin dynamics. In this study, we demonstrate that Arabidopsis ADFs exhibit a moonlighting function in the nucleus, where they interact with transcriptional machinery to regulate the transcriptome during both the resting state and the immune responses. We show that ADF2/3/4 have redundant functions in defense against virulent and avirulent Pseudomonas syringae. Notably, it is nuclear - rather than cytosolic - ADFs that contribute to defense against P. syringae and mediate pro-immune transcription. Mechanistically, we demonstrate that nuclear ADFs interact with transcription factors, histone complexes, and other components of the transcriptional machinery. Specifically, ADF2/3/4 can form a complex with WRKY transcription factors, such as WRKY22/29/48, thereby directly regulating WRKY activity to shape the pro-immune transcriptome. In summary, our study reveals that ADFs moonlight as direct regulators of transcription factors, mediating a broad range of nuclear-cytoplasmic regulation in plant immunity and potentially other biological processes.

Environmental temperature has a huge impact on multiple meiosis processes in flowering plants. Polyploid plants derived from whole genome duplication are believed to have an enhanced abiotic stress tolerance. In this study, the impact of high temperatures on male meiosis in autotetraploid Arabidopsis thaliana was investigated. We found that autotetraploid Columbia (Col-0) plants generate a subpopulation of aberrant meiotic products under normal temperature, which is significantly increased under heat stress. Cytological studies revealed that, as the case in diploid Arabidopsis thaliana, assembly of microtubular cytoskeleton network, pairing and segregation of homologous chromosomes, and meiotic recombination in autotetraploid Arabidopsis are compromised under the high temperatures. Immunostaining of {ramshorn}H2A.X and recombinase DMC1 suggested that heat stress inhibits formation of DNA double-strand breaks; additionally, it specifically destabilizes ASY1 and ASY4, but not SYN1 on chromosomes. The loading defects of ASY1 and ASY4 overlap in the syn1 mutant, which supports that the building of lateral element of synaptonemal complex occurs downstream of a SYN1-ASY4-ASY3 stepwise assembly of axis. Remarkably, heat-induced abnormalities of ASY1 and ASY4 co-localize on chromosomes of both diploid and autotetraploid Arabidopsis, suggesting that high temperatures interfere with ASY1-associated SC via an impacted stability of chromosome axis. Furthermore, ZYP1-dependent transverse filament of SC is disrupted by heat stress. Taken together, these findings suggest that polyploidization negatively contributes to instability of chromosome axis and meiotic recombination in Arabidopsis thaliana under heat stress.

Elevated stress signaling often compromises plant growth by suppressing proliferative and formative divisions at the meristem. Plant Elicitor Peptide 1 (PEP1), an endogenous danger signal triggered by both biotic and abiotic stresses in Arabidopsis thaliana, suppresses proliferative divisions, alters xylem vessel organizations, and disrupts cell-to-cell symplastic connections in the root. To gain an insight into the dynamic molecular framework that modulates root development under elevated danger signals, we performed a time course RNA-sequencing analysis at the root meristem following PEP1 treatment. A series of data analyses revealed that STZ and its homologs are a potential nexus between the stress response and proliferative cell cycle regulation. We observed that STZ differentially controls the cell cycle, cell differentiation, and stress response genes at various tissue layers in the root meristem through various functional, phenotypic, and transcriptomic analyses. Our study further indicated that the expression level of STZ in response to stresses is critically important to enable the growth and defense tradeoff. The findings here provide valuable information about the dynamic gene expression changes upon perceiving danger signals at the root meristem and future engineering schemes to generate stress-resilient plants.

Elucidating regulators and molecular mechanisms underlying gene transcriptional and post-transcriptional co-regulatory network is key to understand plant cold-stress responses. Previous studies were mainly conducted on single species and whether the regulators and mechanisms are conserved across different species remains elusive. Here, we selected three species that diverged at early evolution of rosids (93-115 million years ago) and integrated phylotranscriptome and ChIP/DAP-seq datasets to identify cold-responsive regulators and their regulatory networks. First, we found over ten thousand cold-responsive genes including differentially expressed genes (DEGs) and alternative splicing genes (DASGs) in each species. Among the DEGs, genes encoding a set of transcription factors (TFs) (AP2/ERF, MYB, WRKY, NAC, etc.) and RNA binding proteins (RBPs) (Ribosomal, RRM, DEAD, Helicase_C, etc.) are conserved in cold responses in rosids. Compared to TFs, RBPs show a delayed cold-responsive pattern, likely suggesting a hierarchical regulation of DEGs and DASGs. Between DEGs and DASGs, we identified 259 overlapping DE-DASG orthogroups and interestingly, pathway analysis on each dataset of DEGs, DASGs, and DE-DASGs coincidently shows an enrichment of circadian rhythm. Evidentially, many circadian components are cold-regulated at both transcriptional and post-transcriptional levels. Moreover, we reasoned 226 cold-responsive genes regulated by at least two of five circadian components (CCA1, LHY, RV4, RVE8, and RVE7) in rosids. Finally, we unveiled a conserved hierarchical network in dynamic transcriptional and post-transcriptional regulation of cold-responsive genes launched by circadian components in rosids. Together, our results provide insights into core regulators and mechanisms underlying cold-responsive regulatory network across rosids, despite a long evolutionary history.

Polyploidization is a driving force of wheat evolution and speciation, yet its impact on epigenetic regulation and gene expression remains unclear. Here, we constructed a high-resolution epigenetic landscape across leaves, spikes, and roots of hexaploidy wheat and its tetraploid and diploid relatives. Inter-species stable-expression genes exhibited conserved amino acid sequences under strong purifying selection, while dynamic-expression genes were linked to species-specific adaptation. During hexaploidization, dominant D-subgenome homoeolog expression was suppressed via reduced activating epigenetic signals, converging expression with the A and B subgenomes. Proximal chromatin regions near genes were more stable, whereas distal regions, particularly enhancer-like elements mediated by H3K27ac and H3K4me3, exhibit higher dynamism. Sequence variations in these enhancers lead to differential gene regulation, influencing traits such as spike development. For instance, the two haplotypes of dCRE region of TaDEP-B1 resulted in significant differences in its expression and spikelet numbers. We also observed a coevolution of transcription factors and their binding sites, particularly within the expanded ERF family, which regulates spike morphology. This study highlights the interplay between sequence variation and epigenetic modifications in shaping transcriptional regulation during wheat speciation, offering valuable insights for genetic improvement.

Plants have evolved remarkable diversity in inflorescence architecture. At the center of this diversity lies a meristem maturation program featured by transition of stem cell populations from a vegetative state into a reproductive growth, determining when, where, and how many flowers are produced on inflorescences. Here we identified a new meristem maturation regulator TMF FAMILY MEMBER3 (TFAM3) that encodes an ALOG family transcription factor. Loss of TFAM3 results in early flowering and simplified inflorescences with fewer flowers. Genetic analysis by creating high-order mutants of TFAM3 with three key regulators of tomato shoot meristem maturation, TERMINATING FLOWER (TMF), TMF FAMILY MEMBER1 (TFAM1) and TMF FAMILY MEMBER2 (TFAM2), suggested that they synergistically control flowering transition and inflorescence architecture. The four paralogous ALOG proteins share the prion-like properties and undergo liquid-liquid phase separation in vitro. Strikingly, TMF can recognize cognate TFAM proteins and selectively recruit them into phase separated condensates. Supporting this, they interact with themselves and each other to form biomolecular condensates in the nucleus. Their interaction induces formation of transcriptional condensates that directly repress expression of floral identity gene ANANTHA. Our study revealed a selective-recruitment phase separation mechanism for transcriptional condensation by which plants achieve optimal coordination of functional overlapped paralogs within a protein family to enable precise control of shoot meristem maturation for flowering and production of compound inflorescences.

DNA replication stress threatens genome stability in eukaryotes. The evolutionarily conserved kinase WEE1 is essential for the activation of the replication stress response. Although the polymerase-associated factor 1 complex (PAF1C) is highly conserved in eukaryotes, its role in the DNA replication stress response remains unclear. Here, we show that Arabidopsis PAF1C is essential for replication stress response. PAF1C-deficient mutants exhibit hypersensitivity to hydroxyurea (HU)-induced replication stress. Mechanistically, we uncover a plant-specific regulatory pathway in which WEE1 interacts with and phosphorylates the PAF1 subunit within its unique N-terminal domain, thereby preventing PAF1 polyubiquitination and subsequent proteasomal degradation to ensure PAF1 accumulation under stress. Genetically, overexpression of a phospho-mimetic PAF1 variant suppresses the HU hypersensitivity of wee1, revealing that PAF1 acts downstream of WEE1. However, the WEE1-PAF1 regulatory axis is absent in yeast, indicating its lineage-specific innovation. Further studies reveal that the replication factor C (RFC) complex interacts with and recruits PAF1 to the stalled replication forks. PAF1 then sequentially recruits the E2 ubiquitin-conjugating enzymes UBC1/2 and the E3 ubiquitin ligases HUB1/2 to promote histone H2B monoubiquitination (H2Bub), thereby facilitating replication fork stability. This RFC-dependent recruitment mechanism is conserved in yeast. Collectively, this study suggests that PAF1 regulates replication stress responses by integrating a plant-specific protein stability control mechanism (WEE1-PAF1) with a conserved recruitment mechanism (RFC-PAF1-UBC1/2-HUB1/2), uncovering a novel function of PAF1C and revealing new mechanisms of WEE1 and the RFC complex.

Cryptochromes (CRYs) are photolyase-like blue-light / ultraviolet-A (UV-A) receptors that regulate diverse aspects of plant growth. Maize (Zea mays), a major crop often grown under high UV-B radiation, possesses four copies of CRY. However, it remains unclear whether the multiple copies of CRY in maize have evolved to improve UV tolerance or to acquire new functions. In this study, CRISPR-Cas9-engineered Zmcry mutants were used to investigate the functions of four cryptochromes (ZmCRYs) in maize. The findings revealed that ZmCRYs play a redundant role in mediating blue light signaling and in inhibiting the elongation of the mesocotyl. The results also demonstrated that ZmCRYs mediated blue light-enhanced UV-B stress tolerance in Zea mays by upregulating the expression of genes involved in UV-B stress tolerance-related metabolites such as phenylpropanoid, flavonoid, and fatty acid biosynthesis. Furthermore, blue light was found to influence both the accumulation and composition of epidermal waxes, suggesting that blue light enhances epidermal wax accumulation for UV-B stress tolerance. Additionally, it was discovered that ZmCRY1 directly interacted with GLOSSY2 (GL2) in a blue light dependent manner to mediate blue light promoted C32 aldehyde accumulation, shedding new light on the enigma of aldehyde-forming. These results highlight the critical roles of ZmCRY1s in mediating blue light regulated epidermal wax biosynthesis and UV-B tolerance in Zea mays.

Momilactone A, an important plant labdane-related diterpenoid, functions as a phytoalexin against pathogens and an allelochemical against neighboring plants. The genes involved in biosynthesis of momilactone A are found in clusters, i.e., MABGCs (Momilactone A biosynthetic gene clusters), in the rice and barnyardgrass genomes. How MABGCs originate and evolve is still not clear. Here, we integrated results from comprehensive phylogeny and comparative genomic analyses of the core genes of MABGC-like clusters and MABGCs in 40 monocot plant genomes, providing convincing evidence for the birth and evolution of MABGCs in grass species. The MABGCs found in the PACMAD clade of the core grass lineage (including Panicoideae and Chloridoideae) originated from a MABGC-like cluster in Triticeae (BOP clade) via horizontal gene transfer (HGT) and followed by recruitment of MAS and CYP76L1 genes. The MABGCs in Oryzoideae originated from PACMAD through another HGT event and lost CYP76L1 afterwards. The Oryza MABGC and another Oryza diterpenoid cluster c2BGC are two distinct clusters, with the latter being originated from gene duplication and relocation within Oryzoideae. Further comparison of the expression patterns of the MABGC genes between rice and barnyardgrass in response to pathogen infection and allelopathy provides novel insights into the functional innovation of MABGCs in plants. Our results demonstrate HGT-mediated origination of MABGCs in grass and shed lights into the evolutionary innovation and optimization of plant biosynthetic pathways.

Arabidopsis (Arabidopsis thaliana) is a vital model organism in plant biology and genetics. In the last two decades, researchers have made significant progresses in characterizing the chromatin conformation and epigenomic information within the Arabidopsis genome. This information includes but is not limited to the higher structure of chromosomes, histone modification, DNA methylation, and chromatin accessibility. The results of these studies have provided an additional layer of information that complements the DNA sequence data. However, utilizing such knowledge poses a challenge for certain groups that lack bioinformatics analysts or adequate computing resources. A user-friendly and reproducible platform for accessing this information is urgently needed. In this study, we have developed a comprehensive epigenomic database for Arabidopsis (AraENCODE http://glab.hzau.edu.cn/AraENCODE), which comprises a total of 4,511 data libraries, including published chromatin conformation capture datasets (Hi-C, HiChIP), epigenomic datasets (ChIP-Seq, ATAC-Seq, FAIRE-Seq, MNase-Seq, DNase-Seq, BS-seq), and transcriptome data (RNA-Seq, miRNA-Seq). Furthermore, we have incorporated various existing resources, such as single nucleotide polymorphisms (SNPs), cis-regulatory modules, and multi-omics associations. We aim to provide a novel platform for investigating the regulation of epigenetic and chromatin interactions in Arabidopsis in relation to biological processes.

Polyploidy serves as a major force in plant evolution and domestication of cultivated crops. However, the relationship and underlying mechanism between three-dimensional (3D) chromatin organization and gene expression upon rice genome duplication is largely unknown. Here we compared the 3D chromatin structures between diploid (2C) and autotetraploid (4C) rice by high-throughput chromosome conformation capture analysis, and found that 4C rice presents weakened intra-chromosomal interactions compared to its 2C progenitor. Moreover, we found that changes of 3D chromatin organizations including chromatin compartments, topologically associating domain (TAD) and loops uncouple from gene expression. Moreover, DNA methylations in the regulatory sequences of genes in compartment A/B switched regions and TAD boundaries are not related to their expressions. Importantly, in contrast to that there was no significant difference of methylation levels in TEs in promoters of differentially expressed genes (DEGs) and non-DEGs between 2C and 4C rice, we found that the hypermethylated transposable elements across genes in compartment A/B switched regions and TAD boundaries suppress the expression of these genes. We propose that the rice genome doubling might modulate TE methylation which results in the disconnection between the alteration of 3D chromatin structure and gene expression.

Lateral roots (LRs) are crucial for plants to sense environmental signals in addition to water and nutrient absorption. Auxin is key for LR formation, but the underlying mechanisms are not fully understood. Here we report that Arabidopsis ERF1 inhibits LR emergence by promoting local auxin accumulation with altered distribution and regulating auxin signaling. Loss of ERF1 increases LR density compared with the wild type, whereas ERF1 overexpression causes the opposite phenotype. ERF1 enhances auxin transport by upregulating PIN1 and AUX1, resulting in excessive auxin accumulation in the endodermal, cortical, and epidermal cells surrounding LR primordia. Furthermore, ERF1 represses ARF7 transcription, consequently affecting the expression of cell wall remodeling genes that facilitate LR emergence. Together, our study reveals that ERF1 integrates environmental signals to promote local auxin accumulation with altered distribution and repress ARF7, consequently inhibiting LR emergence in adaptation to fluctuating environments. HighlightsO_LIERF1 functions as a negative regulator of lateral root emergence C_LIO_LIERF1 enhances rootward and shootward auxin transport by directly upregulating the expression of PIN1 and AUX1, resulting in high local auxin accumulation and abnormal auxin distribution in the endodermal, cortical, and epidermal cells overlying lateral root primordia C_LIO_LIERF1 represses the transcription of ARF7 and cell wall remodeling genes in lateral root emergence C_LI

Organelle-derived nuclear DNAs, nuclear plastid DNAs (NUPTs) and nuclear mitochondrial DNAs (NUMTs), have been identified in plants. Most, if not all, genes residing in NUPTs/NUMTs (NUPGs/NUMGs) are known to be inactivated and pseudogenized. However, the role of epigenetic control in silencing NUPGs/NUMGs and the dynamic evolution of NUPTs/NUMTs with respect to organismal phylogeny remain barely explored. Based on the available nuclear and organellar genomic resources of the Triticum/Aegilops complex species, we investigated the evolutionary fates of NUPTs/NUMTs in terms of their epigenetic silencing and their dynamic occurrence rates in the nuclear diploid genomes and allopolyploid subgenomes. NUPTs and NUMTs possessed similar genomic atlas, including preferential integration to the transposable element-rich intergenic regions and generating sequence variations in the nuclear genome. The global transcriptional silencing of NUPGs/NUMGs with disrupted and intact open reading frames can be mainly attributed to their repressive chromatin states, namely high levels of DNA methylation and low levels of active histone modifications. Phylogenomic analyses suggested that the species-specific and gradual accumulation of NUPTs/NUMTs accompanied the speciation processes. Moreover, based on further pan-genomic analyses, we found significant subgenomic asymmetry in the NUPT/NUMT occurrence, which accumulated during allopolyploid wheat evolution. Our findings provide novel insights into the dynamic evolutionary fates of organelle-derived nuclear DNA in plants.

Intracellular Ca2+ controls various cellular functions and local Ca2+ dynamics is tightly regulated upon environmental cues. Maintaining cellular Ca2+ balance is essential for plant survival. Here we report a calcium-dependent protein kinases (CPKs)-mediated signaling pathway, in conjunction with the ER membrane-resident Ca2+-ATPase ECA1, acts influentially for cytosolic Ca2+ homeostasis and osmotic stress tolerance. We show that targeting cytosolic Ca2+ efflux via specific inhibitors or eca1 mutation results in augmented [Ca2+]cyt spikes, elevated cytoplasmic ABA ([ABA]cyt) level and ultimately hypersensitive to osmotic stress. Screening of Arabidopsis CPKs revealed direct binding of CPK2/6/11 to ECA1. Moreover, CPK2/6/11 phosphorylate the N-terminal of ECA1 at Ser5, thereby enhancing its activity for cytosolic Ca2+ efflux into ER and subsequently lower [ABA]cyt. The cumulative effect of ECA1 and CPKs mutation on Arabidopsis plant sensitivity to osmotic stress further illustrates that CPKs/ECA1 acts an intracellular sensory module for plant stress tolerance via regulating [Ca2+]cyt and [ABA]cyt homeostasis. One-sentence summary: CPKs/ECA1 acts an intracellular sensory module for plant osmotic stress tolerance via regulating cytosolic Ca2+ and ABA homeostasis.

Living organisms face threats to genome integrity caused by environmental challenges or metabolic errors in proliferating cells. To avoid the spread of mutations, cell division is temporarily arrested while repair mechanisms deal with DNA lesions. Afterwards, cells either resume division or respond to unsuccessful repair by withdrawing from the cell cycle and undergoing cell differentiation or cell death. How the success rate of DNA repair connects to the execution of cell death remains incompletely known, particularly in plants. Here we provide evidence that the Arabidopsis thaliana RETINOBLASTOMA-RELATED1 (RBR) protein, shown to play structural and transcriptional functions in the DNA damage response (DDR), coordinates these processes in time by successive interactions through its B-pocket sub-domain. Upon DNA damage induction, RBR forms nuclear foci; but the N849F substitution in the B-pocket, which specifically disrupts binding to LXCXE motif-containing proteins, abolishes RBR focus formation and leads to growth arrest. After RBR focus formation, the stress-responsive gene NAC044 arrests cell division. As RBR is released from nuclear foci, it can be bound by the conserved LXCXE motif in NAC044. RBR-mediated cell survival is inhibited by the interaction with NAC044. Disruption of NAC044-RBR interaction impairs the cell death response but is less important for NAC044 mediated growth arrest. Noteworthy, unlike many RBR interactors, NAC044 binds to RBR independent of RBR phosphorylation. Our findings indicate that the availability of the RBR B-pocket to interact with LXCXE-containing proteins couples RBR DNA repair functions and RBR transcriptional functions of in the cell death program.