Overexpression of ZmEXPA5 reduces anthesis-silking interval and increases grain yield under drought and well-watered conditions in maize.

Drought is one of the major abiotic stresses affecting the maize production worldwide. As a cross-pollination crop, maize is sensitive to water stress at flowering stage. Drought at this stage leads to asynchronous development of male and female flower organ and increased interval between anthesis and silking, which finally causes failure of pollination and grain yield loss. In the present study, the expansin gene ZmEXPA5 was cloned and its function in drought tolerance was characterized. An indel variant in promoter of ZmEXPA5 is significantly associated with natural variation in drought-induced anthesis-silking interval. The drought susceptible haplotypes showed lower expression level of ZmEXPA5 than tolerant haplotypes and lost the cis-regulatory activity of ZmDOF29. Increasing ZmEXPA5 expression in transgenic maize decreases anthesis-silking interval and improves grain yield under both drought and well-watered environments. In addition, the expression pattern of ZmEXPA5 was analyzed. These findings provide insights into the genetic basis of drought tolerance and a promising gene for drought improvement in maize breeding. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-023-01432-x.

Identification of a new QTL underlying seminal root number in a maize-teosinte population.

Seminal roots play an important role in acquisition of water and nutrients by maize seedlings. Compared with its teosinte ancestor, maize underwent a change in seminal root number (SRN). Although several key genes controlling SRN have been cloned, identification and utilization of new genes from teosinte would be useful for improving maize root architecture. In this study, a maize-teosinte BC2F6 population containing 206 individuals genotyped by resequencing was used to conduct high-resolution quantitative trait locus (QTL) mapping of SRN. A new major QTL on chromosome 7 (qSRN7) was identified. Differentially expressed genes (DEGs) based on RNA-Seq were identified between two inbred lines with no SRN and multiple SRN at two periods of seminal roots primordia formation. A total of 116 DEGs detected in at least one period were identified within the qSRN7 interval. Three DEGs (Zm00001d021572, Zm00001d021579 and Zm00001d021861) associated with SRN were identified through regional association mapping. When compared with reported domestication-related selective sweeps, Zm00001d021572 was selected during maize domestication. Our findings provide important insights into the genetic basis of SRN and identify a promising candidate gene for further studies on SRN.

Genomic insight into changes of root architecture under drought stress in maize.

Drought stress is a central environmental factor that severely limits maize production worldwide. Root architecture plays an important role in drought tolerance and can be targeted in breeding programmes. Here, we conducted phenotyping of root architecture under different water treatments for 373 maize inbred lines, representative germplasm from both China and the United States in different breeding eras. We found that seminal root length in response to drought stress experienced convergent increase during breeding in both countries. Using a genome-wide association study, we identified a total of 221 associated loci underlying 13 root traits under well-watered and water-stressed conditions. These loci harboured many reported root- and abiotic stress-related genes. Furthermore, a total of 75 strong candidate genes were prioritised by integrating candidate genes associated with seminal root length and differentially expressed genes in seminal root. One of high-confidence candidate genes, ZmCIPK3 was functionally characterised and probably plays a role in enhancing drought tolerance through regulating seminal root growth. This study provides valuable information for genetic improvement of root architecture and drought tolerance in maize.

Phase-separating pyrenoid proteins form complexes in the dilute phase.

While most studies of biomolecular phase separation have focused on the condensed phase, relatively little is known about the dilute phase. Theory suggests that stable complexes form in the dilute phase of two-component phase-separating systems, impacting phase separation; however, these complexes have not been interrogated experimentally. We show that such complexes indeed exist, using an in vitro reconstitution system of a phase-separated organelle, the algal pyrenoid, consisting of purified proteins Rubisco and EPYC1. Applying fluorescence correlation spectroscopy (FCS) to measure diffusion coefficients, we found that complexes form in the dilute phase with or without condensates present. The majority of these complexes contain exactly one Rubisco molecule. Additionally, we developed a simple analytical model which recapitulates experimental findings and provides molecular insights into the dilute phase organization. Thus, our results demonstrate the existence of protein complexes in the dilute phase, which could play important roles in the stability, dynamics, and regulation of condensates.

PIL transcription factors directly interact with SPLs and repress tillering/branching in plants.

Tillering is an important parameter of plant architecture in cereal crops. In this study, we identified the PHYTOCHROME-INTERACTING FACTOR-LIKE (PIL) family transcription factors as new repressors of tillering in cereal crops. Using biochemical and genetic approaches, we explore the roles of TaPIL1 in regulating wheat plant architecture. We found that the PIL protein TaPIL1 controls tiller number in wheat. Overexpression of TaPIL1 reduces wheat tiller number; additionally, overexpression of TaPIL1-SUPERMAN repression domain increases wheat tiller number. Furthermore, we show that TaPIL1 activates the transcriptional expression of wheat TEOSINTE BRANCHED1 (TaTB1); moreover, TaPIL1 physically interacts with wheat SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (TaSPL)3/17, which are activators of TaTB1 transcription. In rice, overexpression and loss-of-function mutations of OsPIL11 reduce or increase tiller number by regulating the expression of OsTB1. In Arabidopsis, we demonstrate that PHYTOCHROME-INTERACTING FACTOR 4 interacts with SPL9 to inhibit shoot branching. This study reveals that PIL family transcription factors directly interact with SPLs and play an important role in repressing tillering/branching in plants.

The blue light receptor CRY1 interacts with GID1 and DELLA proteins to repress gibberellin signaling and plant growth.

Improvements in plant architecture, such as reduced plant height under high-density planting, are important for agricultural production. Light and gibberellin (GA) are essential external and internal cues that affect plant architecture. In this study, we characterize the direct interaction of distinct receptors that link light and GA signaling in Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum L.). We show that the light receptor CRY1 represses GA signaling through interaction with all five DELLA proteins and promotion of RGA protein accumulation in Arabidopsis. Genetic analysis shows that CRY1-mediated growth repression is achieved by means of the DELLA proteins. Interestingly, we find that CRY1 also directly interacts with the GA receptor GID1 to competitively inhibit the GID1-GAI interaction. We also show that overexpression of TaCRY1a reduces plant height and coleoptile growth in wheat and that TaCRY1a interacts with both TaGID1 and Rht1 to competitively attenuate the TaGID1-Rht1 interaction. Based on these findings, we propose that the photoreceptor CRY1 competitively inhibits the GID1-DELLA interaction, thereby stabilizing DELLA proteins and enhancing their repression of plant growth.

The transcription factor TaLAX1 interacts with Q to antagonistically regulate grain threshability and spike morphogenesis in bread wheat.

The domestication gene Q is largely responsible for the widespread cultivation of wheat because it confers multiple domestication traits. However, the underlying molecular mechanisms of how Q regulates these domestication traits remain unclear. In this study, we identify a Q-interacting protein TaLAX1, a basic helix-loop-helix transcription factor, through yeast two-hybrid assays. Using biochemical and genetic approaches, we explore the roles of TaLAX1 in regulating wheat domestication traits. Overexpression of TaLAX1 produces phenotypes, reminiscent of the q allele; loss-of-function Talax1 mutations confer compact spikes, largely similar to the Q-overexpression wheat lines. The two transcription factors TaLAX1 and Q disturb each other's activity to antagonistically regulate the expression of the lignin biosynthesis-related gene TaKNAT7-4D. More interestingly, a natural variation (InDel, +/- TATA), which occurs in the promoter of TaLAX1, is associated with the promoter activity difference between the D subgenome of bread wheat and its ancestor Aegilops tauschii accession T093. This study reveals that the transcription factor TaLAX1 physically interacts with Q to antagonistically regulate wheat domestication traits and a natural variation (InDel, +/- TATA) is associated with the diversification of TaLAX1 promoter activity.

BIC1 acts as a transcriptional coactivator to promote brassinosteroid signaling and plant growth.

The BRASSINAZOLE-RESISTANT 1 (BZR1) transcription factor family plays an essential role in plant brassinosteroid (BR) signaling, but the signaling mechanism through which BZR1 and its homologs cooperate with certain coactivators to facilitate transcription of target genes remains incompletely understood. In this study, we used an efficient protein interaction screening system to identify blue-light inhibitor of cryptochromes 1 (BIC1) as a new BZR1-interacting protein in Arabidopsis thaliana. We show that BIC1 positively regulates BR signaling and acts as a transcriptional coactivator for BZR1-dependent activation of BR-responsive genes. Simultaneously, BIC1 interacts with the transcription factor PIF4 to synergistically and interdependently activate expression of downstream genes including PIF4 itself, and to promote plant growth. Chromatin immunoprecipitation assays demonstrate that BIC1 and BZR1/PIF4 interdependently associate with the promoters of common target genes. In addition, we show that the interaction between BIC1 and BZR1 is evolutionally conserved in the model monocot plant Triticum aestivum (bread wheat). Together, our results reveal mechanistic details of BR signaling mediated by a transcriptional activation module BIC1/BZR1/PIF4 and thus provide new insights into the molecular mechanisms underlying the integration of BR and light signaling in plants.

The structural basis of Rubisco phase separation in the pyrenoid.

Approximately one-third of global CO2 fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO2-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

The HD-ZIP II Transcription Factors Regulate Plant Architecture through the Auxin Pathway.

The homeodomain-leucine zipper (HD-ZIP) family transcription factors play important roles in plant growth and development. However, the underlying mechanisms remain largely unclear. Here we found that ATHB2, encoding a HD-ZIP transcription factor, is an early auxin responsive gene. Phenotypic analyses show that overexpression of ATHB2 impairs plant architecture, including reduced plant height and small leaves, and also reduces auxin response in leaves when grown in soil. Simultaneously, the seedlings with chemical induction of ATHB2 exhibit abnormal root gravitropism, a typical auxin-related phenotype. We further show that the auxin response pattern is altered in roots of the inducible ATHB2 seedlings. Consistently, the transcript levels of some auxin biosynthetic and transport genes are significantly decreased in these transgenic seedlings. Further, protein and promoter sequence analyses in common wheat showed that the HD-ZIP II subfamily transcription factors have highly conserved motifs and most of these encoding gene promoters contain the canonical auxin-responsive elements. Expression analyses confirm that some of these HD-ZIP II genes are indeed regulated by auxin in wheat. Together, our results suggest that the HD-ZIP II subfamily transcription factors regulate plant development possibly through the auxin pathway in plants.

Measuring the predictability of life outcomes with a scientific mass collaboration.

How predictable are life trajectories? We investigated this question with a scientific mass collaboration using the common task method; 160 teams built predictive models for six life outcomes using data from the Fragile Families and Child Wellbeing Study, a high-quality birth cohort study. Despite using a rich dataset and applying machine-learning methods optimized for prediction, the best predictions were not very accurate and were only slightly better than those from a simple benchmark model. Within each outcome, prediction error was strongly associated with the family being predicted and weakly associated with the technique used to generate the prediction. Overall, these results suggest practical limits to the predictability of life outcomes in some settings and illustrate the value of mass collaborations in the social sciences.

Rigidity enhances a magic-number effect in polymer phase separation.

Cells possess non-membrane-bound bodies, many of which are now understood as phase-separated condensates. One class of such condensates is composed of two polymer species, where each consists of repeated binding sites that interact in a one-to-one fashion with the binding sites of the other polymer. Biologically-motivated modeling revealed that phase separation is suppressed by a "magic-number effect" which occurs if the two polymers can form fully-bonded small oligomers by virtue of the number of binding sites in one polymer being an integer multiple of the number of binding sites of the other. Here we use lattice-model simulations and analytical calculations to show that this magic-number effect can be greatly enhanced if one of the polymer species has a rigid shape that allows for multiple distinct bonding conformations. Moreover, if one species is rigid, the effect is robust over a much greater range of relative concentrations of the two species.

Photoexcited phytochrome B interacts with brassinazole resistant 1 to repress brassinosteroid signaling in Arabidopsis.

Photoreceptor phytochrome B (phyB) mediates a variety of light responses in plants. To further elucidate the molecular mechanisms of phyB-regulated hypocotyl elongation, we performed firefly luciferase complementation imaging (LCI) screening for phyB-interacting transcription factors (TFs). LCI assays showed that phyB possibly interacts with brassinazoleresistant 1 (BZR1), BZR2, AUXIN RESPONSE FACTOR 6 (ARF6), and several WRKY DNA-binding TFs in a red light-dependent manner. Furthermore, biochemical assays demonstrated that photoexcited phyB specifically interacts with non-phosphorylated BZR1, the physiologically active form of a master TF in brassinosteroid (BR) signaling, and this interaction can be competitively interfered by phytochrome-interacting factor 4. Furthermore, we showed that phyB can directly interact with the DNA-binding domain of BZR1 and affect the enrichment of BZR1 on the chromatin of target genes. Moreover, our genetic evidence and RNA-seq analysis demonstrated that phyB negatively regulates BR signaling. Together, we revealed that photoexcited phyB directly interacts with the TF BZR1 to repress BR signaling in Arabidopsis.

The Blue-Light Receptor CRY1 Interacts with BZR1 and BIN2 to Modulate the Phosphorylation and Nuclear Function of BZR1 in Repressing BR Signaling in Arabidopsis.

The blue-light receptor cryptochrome 1 (CRY1) primarily mediates blue-light inhibition of hypocotyl elongation in Arabidopsis. However, the underlying mechanisms remain largely elusive. We report here that CRY1 inhibits hypocotyl elongation by repressing brassinosteroid (BR) signaling. A genetic interaction assay reveals the negative regulatory effect of CRY1 on the function of BZR1, a core transcription factor in the BR signaling pathway. We demonstrated that CRY1 interacts with the DNA-binding domain of BZR1 to interfere with the DNA-binding ability of BZR1, and represses its transcriptional activity. Furthermore, we found that CRY1 promotes the phosphorylation of BZR1 and inhibits the nuclear accumulation of BZR1. Interestingly, we discovered that CRY1 interacts with the GSK3-like kinase BIN2 and enhances the interaction of BIN2 and BZR1 in a light-dependent manner. Our findings revealed that CRY1 negatively regulates the function of BZR1 through at least two mechanisms: interfering with the DNA-binding ability of BZR1 and promoting the phosphorylation of BZR1. Therefore, we uncover a novel CRY1-BIN2-BZR1 regulatory module that mediates crosstalk between blue light and BR signaling to coordinate plant growth in Arabidopsis.

Drought-responsive WRKY transcription factor genes TaWRKY1 and TaWRKY33 from wheat confer drought and/or heat resistance in Arabidopsis.

BACKGROUND: Drought stress is one of the major causes of crop loss. WRKY transcription factors, as one of the largest transcription factor families, play important roles in regulation of many plant processes, including drought stress response. However, far less information is available on drought-responsive WRKY genes in wheat (Triticum aestivum L.), one of the three staple food crops. RESULTS: Forty eight putative drought-induced WRKY genes were identified from a comparison between de novo transcriptome sequencing data of wheat without or with drought treatment. TaWRKY1 and TaWRKY33 from WRKY Groups III and II, respectively, were selected for further investigation. Subcellular localization assays revealed that TaWRKY1 and TaWRKY33 were localized in the nuclei in wheat mesophyll protoplasts. Various abiotic stress-related cis-acting elements were observed in the promoters of TaWRKY1 and TaWRKY33. Quantitative real-time PCR (qRT-PCR) analysis showed that TaWRKY1 was slightly up-regulated by high-temperature and abscisic acid (ABA), and down-regulated by low-temperature. TaWRKY33 was involved in high responses to high-temperature, low-temperature, ABA and jasmonic acid methylester (MeJA). Overexpression of TaWRKY1 and TaWRKY33 activated several stress-related downstream genes, increased germination rates, and promoted root growth in Arabidopsis under various stresses. TaWRKY33 transgenic Arabidopsis lines showed lower rates of water loss than TaWRKY1 transgenic Arabidopsis lines and wild type plants during dehydration. Most importantly, TaWRKY33 transgenic lines exhibited enhanced tolerance to heat stress. CONCLUSIONS: The functional roles highlight the importance of WRKYs in stress response.

Foxtail Millet NF-Y Families: Genome-Wide Survey and Evolution Analyses Identified Two Functional Genes Important in Abiotic Stresses.

It was reported that Nuclear Factor Y (NF-Y) genes were involved in abiotic stress in plants. Foxtail millet (Setaria italica), an elite stress tolerant crop, provided an impetus for the investigation of the NF-Y families in abiotic responses. In the present study, a total of 39 NF-Y genes were identified in foxtail millet. Synteny analyses suggested that foxtail millet NF-Y genes had experienced rapid expansion and strong purifying selection during the process of plant evolution. De novo transcriptome assembly of foxtail millet revealed 11 drought up-regulated NF-Y genes. SiNF-YA1 and SiNF-YB8 were highly activated in leaves and/or roots by drought and salt stresses. Abscisic acid (ABA) and H2O2 played positive roles in the induction of SiNF-YA1 and SiNF-YB8 under stress treatments. Transient luciferase (LUC) expression assays revealed that SiNF-YA1 and SiNF-YB8 could activate the LUC gene driven by the tobacco (Nicotiana tobacam) NtERD10, NtLEA5, NtCAT, NtSOD, or NtPOD promoter under normal or stress conditions. Overexpression of SiNF-YA1 enhanced drought and salt tolerance by activating stress-related genes NtERD10 and NtCAT1 and by maintaining relatively stable relative water content (RWC) and contents of chlorophyll, superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and malondialdehyde (MDA) in transgenic lines under stresses. SiNF-YB8 regulated expression of NtSOD, NtPOD, NtLEA5, and NtERD10 and conferred relatively high RWC and chlorophyll contents and low MDA content, resulting in drought and osmotic tolerance in transgenic lines under stresses. Therefore, SiNF-YA1 and SiNF-YB8 could activate stress-related genes and improve physiological traits, resulting in tolerance to abiotic stresses in plants. All these results will facilitate functional characterization of foxtail millet NF-Ys in future studies.

Genome-wide analysis of the Hsf family in soybean and functional identification of GmHsf-34 involvement in drought and heat stresses.

BACKGROUND: High temperature affects organism growth and metabolic activity. Heat shock transcription factors (Hsfs) are key regulators in heat shock response in eukaryotes and prokaryotes. Under high temperature conditions, Hsfs activate heat shock proteins (Hsps) by combining with heat stress elements (HSEs) in their promoters, leading to defense of heat stress. Since the first plant Hsf gene was identified in tomato, several plant Hsf family genes have been thoroughly characterized. Although soybean (Glycine max), an important oilseed crops, genome sequences have been available, the Hsf family genes in soybean have not been characterized accurately. RESULT: We analyzed the Hsf genetic structures and protein function domains using the GSDS, Pfam, SMART, PredictNLS, and NetNES online tools. The genome scanning of dicots (soybean and Arabidopsis) and monocots (rice and maize) revealed that the whole-genome replication occurred twice in soybean evolution. The plant Hsfs were classified into 3 classes and 16 subclasses according to protein structure domains. The A8 and B3 subclasses existed only in dicots and the A9 and C2 occurred only in monocots. Thirty eight soybean Hsfs were systematically identified and grouped into 3 classes and 12 subclasses, and located on 15 soybean chromosomes. The promoter regions of the soybean Hsfs contained cis-elements that likely participate in drought, low temperature, and ABA stress responses. There were large differences among Hsfs based on transcriptional levels under the stress conditions. The transcriptional levels of the A1 and A2 subclass genes were extraordinarily high. In addition, differences in the expression levels occurred for each gene in the different organs and at the different developmental stages. Several genes were chosen to determine their subcellular localizations and functions. The subcellular localization results revealed that GmHsf-04, GmHsf-33, and GmHsf-34 were located in the nucleus. Overexpression of the GmHsf-34 gene improved the tolerances to drought and heat stresses in Arabidopsis plants. CONCLUSIONS: This present investigation of the quantity, structural features, expression characteristics, subcellular localizations, and functional roles provides a scientific basis for further research on soybean Hsf functions.