Higher Order Mutants Research Articles

Sextuple knockouts of a highly conserved and coexpressed AUXIN/INDOLE-3-ACETIC ACID gene set confer shade avoidance-like responses in Arabidopsis.

AUXIN/INDOLE-3-ACETIC ACIDs are transcriptional repressors for auxin signalling. Aux/IAAs of Arabidopsis thaliana display some functional redundancy. The IAA3/SHY2 clade (IAA1, IAA2, IAA3and IAA4) show strong sequence similarity, but no higher-order mutants have been reported. Here, through CRISPR/Cas9 genome editing, we generated loss-of-function iaa1/2/3/4 mutants. The quadruple mutants only exhibited a weak phenotype. Thus, we additionally knocked out IAA7/AXR2 and IAA16, which are coexpressed with IAA1/2/3/4. Remarkably, under white light control conditions, the iaa1/2/3/4/7/16 mutants exhibited a shade avoidance-like phenotype with over-elongated hypocotyls and petioles and hyponastic leaves. The sextuple mutants were highly sensitive to low light intensity, and the hypocotyl cells of the mutants were excessively elongated. Transcriptome profiling and qRT-PCR analyses revealed that the sextuple mutation upregulated IAA19/MSG2 and IAA29, two shared shade/auxin signalling targets. Besides, genes encoding cell wall-remodelling proteins and shade-responsive transcription regulators were upregulated. Using dual-luciferase reporter assays, we verified that IAA2/IAA7 targeted the promoters of cell wall-remodelling genes to inhibit their transcription. Our work indicates that the IAA1/2/3/4/7/16 gene set is required for the optimal integration of auxin and shade signalling. The mutants generated here should be valuable for exploring the complex interactions among signal sensors, transcription activatorsand transcription repressors during hormone/environmental responses.

Three Arabidopsis UMP kinases have different roles in pyrimidine nucleotide biosynthesis and (deoxy)CMP salvage.

Pyrimidine nucleotide monophosphate biosynthesis ends in the cytosol with uridine monophosphate (UMP). UMP phosphorylation to uridine diphosphate (UDP) by UMP KINASEs (UMKs) is required for the generation of all pyrimidine (deoxy)nucleoside triphosphates as building blocks for nucleic acids and central metabolites like UDP-glucose. The Arabidopsis (Arabidopsis thaliana) genome encodes five UMKs and three belong to the AMP KINASE (AMK)-like UMKs, which were characterized to elucidate their contribution to pyrimidine metabolism. Mitochondrial UMK2 and cytosolic UMK3 are evolutionarily conserved, whereas cytosolic UMK1 is specific to the Brassicaceae. In vitro, all UMKs can phosphorylate UMP, cytidine monophosphate (CMP) and deoxycytidine monophosphate (dCMP), but with different efficiencies. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9)-induced null mutants were generated for UMK1 and UMK2, but not for UMK3, since frameshift alleles were lethal for germline cells. However, a mutant with diminished UMK3 activity showing reduced growth was obtained. Metabolome analyses of germinating seeds and adult plants of single- and higher-order mutants revealed that UMK3 plays an indispensable role in the biosynthesis of all pyrimidine (deoxy)nucleotides and UDP-sugars, while UMK2 is important for dCMP recycling that contributes to mitochondrial DNA stability. UMK1 is primarily involved in CMP recycling. We discuss the specific roles of these UMKs referring also to the regulation of pyrimidine nucleoside triphosphate synthesis.

Evolutionary conservation of receptor compensation for stem cell homeostasis in Solanaceae plants.

Stem cell homeostasis is pivotal for continuous and programmed formation of organs in plants. The precise control of meristem proliferation is mediated by the evolutionarily conserved signaling that encompasses complex interactions among multiple peptide ligands and their receptor-like kinases. Here, we identified compensation mechanisms involving the CLAVATA1 (CLV1) receptor and its paralogs, BARELY ANY MERISTEMs (BAMs), for stem cell proliferation in two Solanaceae species, tomato and groundcherry. Genetic analyses of higher-order mutants deficient in multiple receptor genes, generated via CRISPR-Cas9 genome editing, reveal that tomato SlBAM1 and SlBAM2 compensate for slclv1 mutations. Unlike the compensatory responses between orthologous receptors observed in Arabidopsis, tomato slclv1 mutations do not trigger transcriptional upregulation of four SlBAM genes. The compensation mechanisms within receptors are also conserved in groundcherry, and critical amino acid residues of the receptors associated with the physical interaction with peptide ligands are highly conserved in Solanaceae plants. Our findings demonstrate that the evolutionary conservation of both compensation mechanisms and critical coding sequences between receptor-like kinases provides a strong buffering capacity during stem cell homeostasis in tomato and groundcherry.

Contributions of lignification, tissue arrangement patterns, and cross-sectional area to whole-stem mechanical properties in Arabidopsis thaliana.

Plant cells withstand mechanical stress originating from turgor pressure by robustly maintaining the mechanical properties of the cell wall. This applies at the organ scale as well; many plant stems act as pressurized cylinders, where the epidermis is under tension and inner tissues are under compression. The clavata3 de-etiolated3 (clv3-8 det3-1) double mutant of Arabidopsis thaliana displays cracks in its stems because of a conflict between the mechanical properties of the weak epidermis and over-proliferation of inner stem tissues. In this work, we conducted three-point bending tests on various Arabidopsis thaliana mutants, including those displaying the stem cracking phenotype, to examine the differences in their mechanical properties. The clv3-8 det3-1 double mutant exhibited reduced stem stiffness, consistent with reduced differentiation due to the clv3-8 mutation. Yet, in clv3-8, stem cross-sectionalarea was increased associating with the increase in vascular bundle number, and stem cross-sections displayed various shapes. To uncouple the contribution of geometry and cell-wall differentiation to the mechanical properties of the whole stems, we tested the contribution of lignified fibers to stem stiffness. In order to suppress lignin deposition in stems genetically, we generated multiple higher-order mutants by crossing clv3-8 and/or det3-1 with nst1-1 nst3-1, in which lignin deposition is suppressed. Stem stiffness was reduced markedly in all nst1-1 nst3-1 mutant backgrounds. Overall, our results suggest that stem stiffness relies on the presence of differentiated, lignified, fiber tissue as well as on the alignment or spatial distribution of vascular bundles within the stem organ.

Diverse INOSITOL PHOSPHORYLCERAMIDE SYNTHASE mutant alleles of Physcomitrium patens offer new insight into complex sphingolipid metabolism.

Sphingolipids are widespread, abundant, and essential lipids in plants and in other eukaryotes. Glycosyl inositol phosphorylceramides (GIPCs) are the most abundant class of plant sphingolipids, and are enriched in the plasma membrane of plant cells. They have been difficult to study due to lethal or pleiotropic mutant phenotypes. To overcome this, we developed a CRISPR/Cas9-based method for generating multiple and varied knockdown and knockout populations of mutants in a given gene of interest in the model moss Physcomitrium patens. This system is uniquely convenient due to the predominantly haploid state of the Physcomitrium life cycle, and totipotency of Physcomitrium protoplasts used for transformation. We used this approach to target the INOSITOL PHOSPHORYLCERAMIDE SYNTHASE (IPCS) gene family, which catalyzes the first, committed step in the synthesis of GIPCs. We isolated knockout single mutants and knockdown higher-order mutants showing a spectrum of deficiencies in GIPC content. Remarkably, we also identified two mutant alleles accumulating inositol phosphorylceramides, the direct products of IPCS activity, and provide our best explanation for this unexpected phenotype. Our approach is broadly applicable for studying essential genes and gene families, and for obtaining unusual lesions within a gene of interest.

Hybridizing genetic algorithm and harmony search for higher-order mutation testing

Mutation testing can evaluate the quality of the test inputs, generate test data, and simulate any test coverage criterion. Genetic algorithms and harmony search have been applied to reduce the cost of generating test inputs. Although hybridizing search algorithms enhances the efficiency of searching the solution domain, there is a shortage of applying the hybrid search techniques in mutation testing. This paper merges the genetic and harmony search algorithms to effectively generate test data to kill higher-order mutants. In addition, the performance of the proposed technique will be evaluated and compared with a stand-alone genetic algorithm and a stand-alone harmony search algorithm through an empirical study using a set of benchmark programs. The experimental study shows that the proposed technique outperformed the compared algorithms, reaching a higher killing ratio, where the proposed approach kills 92.8% of higher-order mutants for all tested programs. In comparison, GA kills 88.7%, and HA kills 86.6%. Besides, the proposed algorithm overcame the compared algorithm in reaching a targeted killing ratio faster than the compared algorithms. HGA reduced the execution time for each program with a reduction ratio ranging from 58.9% to 89.8%.

Delta4Ms: Improving mutation‐based fault localization by eliminating mutant bias

AbstractFault localization is a complex, costly and time‐consuming task in software debugging. Numerous automated techniques have been developed to expedite this process. Mutation‐based fault localization (MBFL) is one of the most widely studied techniques which uses mutation analysis to generate mutants for revealing potential faults in the program. However, our theoretical analysis exposes an inherent conflict between the fundamental assumption and the essential meaning of existing MBFL suspiciousness. This conflict is caused by mutant bias. Intuitively, the suspiciousness can be corrected by eliminating the mutant bias for more accurately measuring the faulty probability of the corresponding mutant statement. In this paper, we introduce Delta4Ms, a fault localization approach designed to eliminate mutant bias. Delta4Ms integrates the principles of signal theory, modelling the actual suspiciousness and mutant bias as the desired and false signal components, respectively. Based on theoretical derivation, the average suspiciousness of mutants serves as an estimate of mutant bias. Delta4Ms effectively mitigates mutant bias, extracting the desired signal and yielding corrected suspiciousness for fault localization. To precisely estimate mutant bias, higher order mutants (HOMs) are incorporated. We conduct an extensive experimental evaluation of Delta4Ms on 320 real‐fault programs from Codeflaws. The results indicate that our model significantly outperforms existing SBFL and MBFL techniques, showing a considerable improvement in fault localization effectiveness. We further assessed the robustness of Delta4Ms by examining different HOM ratios and HOM generation strategies. Moreover, Delta4Ms achieves a substantial reduction in mutation execution cost and minimal accuracy loss through the implementation of test case reduction. Finally, we perform preliminary experiments on 15 real‐fault programs from the Defects4J benchmark to assess the generalization of the model's fault localization effectiveness.

Do we need high-order mutation in fault-based Boolean-specification testing?

Fault-based Boolean-specification testing is an essential weak mutation testing technique since the executing paths of the programs are usually dependent on those Boolean expressions in predicate statements. In mutation testing, the coupling effect hypothesis assumes that a test set that detects all the simple faults in the program can detect a high percentage of complex faults. Such a hypothesis suggests that we do not need to perform high-order mutation testing in practice. Many empirical results have shown the existence of the coupling effect. However, as fault-based Boolean-specification testing is mainly used in safety-critical scenarios, the question of whether high-order mutation testing is necessary for Boolean-specifications needs to be answered more strictly. To answer whether we need high-order mutation testing in fault-based Boolean-specification testing, we conduct an empirical study on general-form Boolean expressions. Experimental results show us that: (1) Generally, it is slightly easier to kill high-order mutants (HOMs) than first-order mutants (FOMs) since fault detection probabilities for complex faults (HOMs) are marginally higher than that for simple faults (FOMs) for almost all the cases. (2) However, for any fault types in Boolean-specifications, the coupling effect hypothesis is not guaranteed to hold with absolute certainty. (3) When HOMs involve the expression negation fault (ENF) or the operator reference fault (ORF), the percent of killed HOMs is lower. Such results suggest that, if the testing resource is sufficient, high-order mutation testing could be carried out on fault types ENF and ORF in fault-based Boolean-specification testing.

Subtilase-mediated biogenesis of the expanded family of SERINE RICH ENDOGENOUS PEPTIDES.

Plant signalling peptides are typically released from larger precursors by proteolytic cleavage to regulate plant growth, development and stress responses. Recent studies reported the characterization of a divergent family of Brassicaceae-specific peptides, SERINE RICH ENDOGENOUS PEPTIDES (SCOOPs), and their perception by the leucine-rich repeat receptor kinase MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2). Here, we reveal that the SCOOP family is highly expanded, containing at least 50 members in the Columbia-0 reference Arabidopsis thaliana genome. Notably, perception of these peptides is strictly MIK2-dependent. How bioactive SCOOP peptides are produced, and to what extent their perception is responsible for the multiple physiological roles associated with MIK2 are currently unclear. Using N-terminomics, we validate the N-terminal cleavage site of representative PROSCOOPs. The cleavage sites are determined by conserved motifs upstream of the minimal SCOOP bioactive epitope. We identified subtilases necessary and sufficient to process PROSCOOP peptides at conserved cleavage motifs. Mutation of these subtilases, or their recognition motifs, suppressed PROSCOOP cleavage and associated overexpression phenotypes. Furthermore, we show that higher-order mutants of these subtilases show phenotypes reminiscent of mik2 null mutant plants, consistent with impaired PROSCOOP biogenesis, and demonstrating biological relevance of SCOOP perception by MIK2. Together, this work provides insights into the molecular mechanisms underlying the functions of the recently identified SCOOP peptides and their receptor MIK2.

Mitochondrial VOLTAGE-DEPENDENT ANION CHANNEL 3 regulates stomatal closure by abscisic acid signaling.

In Arabidopsis (Arabidopsis thaliana), stomatal closure mediated by abscisic acid (ABA) is redundantly controlled by ABA receptor family proteins (PYRABACTIN RESISTANCE 1 [PYR1]/PYR1-LIKE [PYLs]) and subclass III SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASES 2 (SnRK2s). Among these proteins, the roles of PYR1, PYL2, and SnRK2.6 are more dominant. A recent discovery showed that ABA-induced accumulation of reactive oxygen species (ROS) in mitochondria promotes stomatal closure. By analyzing stomatal movements in an array of single and higher order mutants, we revealed that the mitochondrial protein VOLTAGE-DEPENDENT ANION CHANNEL 3 (VDAC3) jointly regulates ABA-mediated stomatal closure with a specialized set of PYLs and SnRK2s by affecting cellular and mitochondrial ROS accumulation. VDAC3 interacted with 9 PYLs and all 3 subclass III SnRK2s. Single mutation in VDAC3, PYLs (except PYR1 and PYL2), or SnRK2.2/2.3 had little effect on ABA-mediated stomatal closure. However, knocking out PYR1, PYL1/2/4/8, or SnRK2.2/2.3 in vdac3 mutants resulted in significantly delayed or attenuated ABA-mediated stomatal closure, despite the presence of other PYLs or SnRK2s conferring redundant functions. We found that cellular and mitochondrial accumulation of ROS induced by ABA was altered in vdac3pyl1 mutants. Moreover, H2O2 treatment restored ABA-induced stomatal closure in mutants with decreased stomatal sensitivity to ABA. Our work reveals that VDAC3 ensures redundant control of ABA-mediated stomatal closure by canonical ABA signaling components.

Endosperm cell death promoted by NAC transcription factors facilitates embryo invasion in Arabidopsis

In flowering plants, two fertilization products develop within the limited space of the seed: the embryo and the surrounding nutritive endosperm. The final size of the endosperm is modulated by the degree of embryo growth. In Arabidopsis thaliana, the endosperm expands rapidly after fertilization, but later gets invaded by the embryo that occupies most of the seed volume at maturity, surrounded by a single remaining aleurone-like endosperm layer.1,2,3,4 Embryo invasion is facilitated by the endosperm-expressed bHLH-type transcription factor ZHOUPI, which promotes weakening of endosperm cell walls.5,6 Endosperm elimination in zou mutants is delayed, and embryo growth is severely affected; the endosperm finally collapses around the dwarf embryo, causing the shriveled appearance of mature zou seeds.5,6,7 However, whether ZHOUPI facilitates mechanical endosperm destruction by the invading embryo or whether an active programmed cell death (PCD) process causes endosperm elimination has been subject to debate.2,8 Here we show that developmental PCD controlled by multiple NAC transcription factors in the embryo-adjacent endosperm promotes gradual endosperm elimination. Misexpressing the NAC transcription factor KIRA1 in the entire endosperm caused total endosperm elimination, generating aleurone-less mature seeds. Conversely, dominant and recessive higher-order NAC mutants led to delayed endosperm elimination and impaired cell corpse clearance. Promoting PCD in the zhoupi mutant partially rescued its embryo growth defects, while the endosperm in a zhoupi nac higher-order mutant persisted until seed desiccation. These data suggest that a combination of cell wall weakening and PCD jointly facilitates embryo invasion by an active auto-elimination of endosperm cells.

Combining multiplex gene editing and doubled haploid technology in maize.

A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.

Deep Mutational Scanning of an Oxygen-Independent Fluorescent Protein CreiLOV for Comprehensive Profiling of Mutational and Epistatic Effects.

Oxygen-independent, flavin mononucleotide-based fluorescent proteins (FbFPs) are promising alternatives to green fluorescent protein in anaerobic contexts. Deep mutational scanning performs systematic profiling of protein sequence-function relationships but has not been applied to FbFPs. Focusing on CreiLOV from Chlamydomonas reinhardtii, we created and analyzed two comprehensive mutant collections: (1) single-residue, site-saturation mutagenesis libraries covering all 118 residues; and (2) a full combinatorial metagenesis library among 20 mutations at 15 residues, where mutation and residue selection was based on single-site mutagenesis results. Notably, the second type of library is indispensable to study higher-order epistasis but underrepresented in the literature. Using optimized FACS-seq assays, 2,185 (>92.5%) out of 2,360 possible single-site mutants and 165,428 (>89.7%) out of 184,320 possible combinatorial mutants were reliably assigned with fitness values. We constructed statistical and machine-learning models to analyze the CreiLOV data set, enabling accurate fitness prediction of higher-order mutants using lower-order mutagenesis data. In addition, we successfully isolated CreiLOV variants with improved fluorescence quantum yield and thermostability. This work provides new empirical data and design rules to engineer combinatorial protein variants.

Optimizing Antibody-Antigen Binding Affinities with the ADAPT Platform.

The ADAPT (Assisted Design of Antibody and Protein Therapeutics) platform guides the selection of mutants that improve/modulate the affinity of antibodies and other biologics. Predicted affinities are based on a consensus z-score from three scoring functions. Computational predictions are interleaved with experimental validation, significantly enhancing the robustness of the design and selection of mutants. A key step is an initial exhaustive virtual single-mutant scan that identifies hot spots and the mutations predicted to improve affinity. A small number of proposed single mutants are then produced and assayed. Only the validated single mutants (i.e., having improved affinity) are used to design double and higher-order mutants in subsequent rounds of design, avoiding the combinatorial explosion that arises from random mutagenesis. Typically, with a total of about 30-50 designed single, double, and triple mutants, affinity improvements of 10- to 100-fold are obtained.

GaSubtle: A New Genetic Algorithm for Generating Subtle Higher-Order Mutants

Mutation testing is an effective, yet costly, testing approach, as it requires generating and running large numbers of faulty programs, called mutants. Mutation testing also suffers from a fundamental problem, which is having a large percentage of equivalent mutants. These are mutants that produce the same output as the original program, and therefore, cannot be detected. Higher-order mutation is a promising approach that can produce hard-to-detect faulty programs called subtle mutants, with a low percentage of equivalent mutants. Subtle higher-order mutants contribute a small set of the large space of mutants which grows even larger as the order of mutation becomes higher. In this paper, we developed a genetic algorithm for finding subtle higher-order mutants. The proposed approach uses a new mechanism in the crossover phase and uses five selection techniques to select mutants that go to the next generation in the genetic algorithm. We implemented a tool, called GaSubtle that automates the process of creating subtle mutants. We evaluated the proposed approach by using 10 subject programs. Our evaluation shows that the proposed crossover generates more subtle mutants than the technique used in a previous genetic algorithm with less execution time. Results vary on the selection strategies, suggesting a dependency relation with the tested code.

Can Higher-Order Mutants Improve the Performance of Mutation-Based Fault Localization?

First-order mutants (FOMs) have been widely used in mutation-based fault localization (MBFL) approaches and have achieved promising results in single-fault localization scenarios (SFL-scenario). Higher-order mutants (HOMs) are proposed to simulate complex faults and can be applied in MBFL theoretically for multiple-fault localization scenarios (MFL-scenario). However, whether HOMs can improve MBFL’s performance is not investigated and the effectiveness is not thoroughly evaluated. In this empirical study, we investigate the impact of HOMs on the performance of MBFL in SFL-scenario and MFL-scenario. The experiments on two real-world benchmarks reveal that 1) 2-HOMs can help improve the MBFL performance in SFL-scenarios; 2) in MFL-scenarios, both 2-HOMs and 3-HOMs can achieve better performance than FOMs; and 3) huge computational cost cannot be ignored in the practice of HOMs. Therefore, effective methods to reduce the number of HOMs for future MBFL studies should be considered.

An Empirical Study on Higher-Order Mutation-Based Fault Localization

Fault localization is one of the most expensive activities in software debugging. Mutation-based fault localization (MBFL) is a commonly studied technique that applied mutation analysis to find the location of faults in the programs. Previous studies showed that MBFL adopted First-Order-Mutants (FOMs) that could achieve promising results in single-fault localization, but it did not perform well in multiple-fault localization. Recently, Higher-Order-Mutants (HOMs) were proposed for modeling complex faults but whether HOMs can help in fault localization is still unknown. In this paper, we investigate the performance of MBFL with FOMs and HOMs on single- and multiple-fault localization. Moreover, to study the characteristics of HOMs, we divide HOMs into three groups (i.e. Accurate HOMs, Partially accurate HOMs, and Inaccurate HOMs) by considering different mutation locations. Based on the empirical results on 186 versions of six real-world programs, we find that (1) In single-fault localization, FOMs can achieve better performance than HOMs. (2) However, in multiple-fault localization, HOMs (2-HOMs) localize more faults than FOMs. (3) Furthermore, different types of HOMs have different fault localization effectiveness, where Accurate HOMs outperform the other two HOMs categories. Therefore, the researchers should propose methods to find HOMs more useful for fault localization.

HOTFUZ: Cost‐effective higher‐order mutation‐based fault localization

AbstractFault localization techniques are used to deduce the exact source of a failure from a set of failure indications while debugging software and play a crucial role in improving software quality. Mutation‐based fault localization (MBFL) techniques are proposed to localize faults at a finer granularity and with higher accuracy than traditional fault localization techniques. Despite the technique's effectiveness, the immense cost of mutation analysis hinders MBFL's practical application in the industry. Various mutation alternative strategies are utilized to lower the cost of MBFL, but they sacrifice the accuracy of localization results. Higher‐order mutation testing was proposed to search for valuable mutants that drive testing harder and reduce the overall test effort. However, higher‐order mutants (HOMs) never have been used to address the cost problem of MBFL to the extent of our knowledge. This paper proposes a novel, cost‐effective MBFL technique called HOTFUZ, Higher‐Order muTation‐based FaUlt localiZation, that employs HOMs to reduce the cost while minimizing the accuracy degradation. HOTFUZ combines mutants of a program under test into HOMs to decrease the number of mutants by more than half, depending on the order of HOMs. An experimental study is conducted using 65 real‐world faults of CoREBench to assess the proposed approach's cost‐effectiveness. The experimental results show that HOTFUZ outperforms the extant mutation alternative strategies by localizing faults more accurately using the same number of mutants executed. HOTFUZ has three main benefits over existing mutant reduction techniques for MBFL: (a) It keeps the advantage of using the whole set of mutation operators; (b) it does not discard generated mutants randomly for the sake of efficiency; and, finally, (c) it significantly decreases the proportion of equivalent mutants.

Raf-like kinases and receptor-like (pseudo)kinase GHR1 are required for stomatal vapor pressure difference response.

Stomatal pores close rapidly in response to low-air-humidity-induced leaf-to-air vapor pressure difference (VPD) increases, thereby reducing excessive water loss. The hydroactive signal-transduction mechanisms mediating high VPD-induced stomatal closure remain largely unknown. The kinetics of stomatal high-VPD responses were investigated by using time-resolved gas-exchange analyses of higher-order mutants in guard-cell signal-transduction branches. We show that the slow-type anion channel SLAC1 plays a relatively more substantial role than the rapid-type anion channel ALMT12/QUAC1 in stomatal VPD signaling. VPD-induced stomatal closure is not affected in mpk12/mpk4GC double mutants that completely disrupt stomatal CO2 signaling, indicating that VPD signaling is independent of the early CO2 signal-transduction pathway. Calcium imaging shows that osmotic stress causes cytoplasmic Ca2+ transients in guard cells. Nevertheless, osca1-2/1.3/2.2/2.3/3.1 Ca2+-permeable channel quintuple, osca1.3/1.7-channel double, cngc5/6-channel double, cngc20-channel single, cngc19/20crispr-channel double, glr3.2/3.3-channel double, cpk-kinase quintuple, cbl1/4/5/8/9 quintuple, and cbl2/3rf double mutants showed wild-type-like stomatal VPD responses. A B3-family Raf-like mitogen-activated protein (MAP)-kinase kinase kinase, M3Kδ5/RAF6, activates the OST1/SnRK2.6 kinase in plant cells. Interestingly, B3 Raf-kinase m3kδ5 and m3kδ1/δ5/δ6/δ7 (raf3/6/5/4) quadruple mutants, but not a 14-gene raf-kinase mutant including osmotic stress-linked B4-family Raf-kinases, exhibited slowed high-VPD responses, suggesting that B3-family Raf-kinases play an important role in stomatal VPD signaling. Moreover, high VPD-induced stomatal closure was impaired in receptor-like pseudokinase GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) mutant alleles. Notably, the classical transient "wrong-way" VPD response was absent in ghr1 mutant alleles. These findings reveal genes and signaling mechanisms in the elusive high VPD-induced stomatal closing response pathway.

Redundant roles of four ZIP family members in zinc homeostasis and seed development in Arabidopsis thaliana.

Zinc (Zn) is essential for normal plant growth and development. The Zn-regulated transporter, iron-regulated transporter (IRT)-like protein (ZIP) family members are involved in Zn transport and cellular Zn homeostasis throughout the domains of life. In this study, we have characterized four ZIP transporters from Arabidopsis thaliana (IRT3, ZIP4, ZIP6, and ZIP9) to better understand their functional roles. The four ZIP proteins can restore the growth defect of a yeast Zn uptake mutant and are upregulated under Zn deficiency. Single and double mutants show no phenotypes under Zn-sufficient or Zn-limited growth conditions. In contrast, triple and quadruple mutants show impaired growth irrespective of external Zn supply due to reduced Zn translocation from root to shoot. All four ZIP genes are highly expressed during seed development, and siliques from all single and higher-order mutants exhibited an increased number of abnormal seeds and decreased Zn levels in mature seeds relative to wild type. The seed phenotypes could be reversed by supplementing the soil with Zn. Our data demonstrate that IRT3, ZIP4, ZIP6, and ZIP9 function redundantly in maintaining Zn homeostasis and seed development in A. thaliana.