Stalks and roots are the main battlefield for the coevolution between maize and Fusarium verticillioides.

Fusarium species are the dominant cause of maize ear rot, but they also inflict serious damage to the roots and stalks. Theoretically, the organ where the host interacts with the pathogen most frequently should exhibit the highest degree of symptom-genotype correlation. Because that symptom-genotype correlation is an indicator reflecting the degree of coevolution between pathogen and its hosts. We wonder which organ is the main battlefield for the antagonism between maize and Fusarium. For this purpose, 43 isolates of Fusarium were isolated from infected maize ears. Fusarium verticillioides and F. graminearum are the two dominant pathogens, accounting for 44% and 30%, respectively. Furthermore, 14 elite maize inbreds were exposed to 43 Fusarium isolates and the symptoms of ear rot, stalk rot and root rot were investigated. In general, symptoms caused by F. graminearum were significantly more severe than those caused by other Fusarium species. Surprisingly, the genotype of F. verticillioides showed a strong correlation with stalk and root rot, but not with ear rot. Accordingly, our study may provide the first evidence that the stalk and root of maize, rather than the ear, is the main battlefield for the coevolution between maize and F. verticillioides.

Drought-induced circular RNAs in maize roots: Separating signal from noise.

Circular RNAs (circRNAs) play an important role in diverse biological processes; however, their origin and functions, especially in plants, remain largely unclear. Here, we used 2 maize (Zea mays) inbred lines, as well as 14 of their derivative recombination inbred lines with different drought sensitivity, to systematically characterize 8,790 circRNAs in maize roots under well-watered (WW) and water-stress (WS) conditions. We found that a diverse set of circRNAs expressed at significantly higher levels under WS. Enhanced expression of circRNAs was associated with longer flanking introns and an enrichment of long interspersed nuclear element retrotransposable elements. The epigenetic marks found at the back-splicing junctions of circRNA-producing genes were markedly different from canonical splicing, characterized by increased levels of H3K36me3/H3K4me1, as well as decreased levels of H3K9Ac/H3K27Ac. We found that genes expressing circRNAs are subject to relaxed selection. The significant enrichment of trait-associated sites along their genic regions suggested that genes giving rise to circRNAs were associated with plant survival rate under drought stress, implying that circRNAs play roles in plant drought responses. Furthermore, we found that overexpression of circMED16, one of the drought-responsive circRNAs, enhances drought tolerance in Arabidopsis (Arabidopsis thaliana). Our results provide a framework for understanding the intricate interplay of epigenetic modifications and how they contribute to the fine-tuning of circRNA expression under drought stress.

Profiling the selected hotspots for ear traits in two maize-teosinte populations.

KEY MESSAGE: Eight selected hotspots related to ear traits were identified from two maize-teosinte populations. Throughout the history of maize cultivation, ear-related traits have been selected. However, little is known about the specific genes involved in shaping these traits from their origins in the wild progenitor, teosinte, to the characteristics observed in modern maize. In this study, five ear traits (kernel row number [KRN], ear length [EL], kernel number per row [KNR], cob diameter [CD], and ear diameter [ED]) were investigated, and eight quantitative trait loci (QTL) hotspots were identified in two maize-teosinte populations. Notably, our findings revealed a significant enrichment of genes showing a selection signature and expressed in the ear in qbdCD1.1, qbdCD5.1, qbpCD2.1, qbdED1.1, qbpEL1.1, qbpEL5.1, qbdKNR1.1, and qbdKNR10.1, suggesting that these eight QTL are selected hotspots involved in shaping the maize ear. By combining the results of the QTL analysis with data from previous genome-wide association study (GWAS) involving two natural panels, we identified eight candidate selected genes related to KRN, KNR, CD, and ED. Among these, considering their expression pattern and sequence variation, Zm00001d025111, encoding a WD40/YVTN protein, was proposed as a positive regulator of KNR. This study presents a framework for understanding the genomic distribution of selected loci crucial in determining ear-related traits.

ZmARF1 positively regulates low phosphorus stress tolerance via modulating lateral root development in maize.

Phosphorus (P) deficiency is one of the most critical factors for plant growth and productivity, including its inhibition of lateral root initiation. Auxin response factors (ARFs) play crucial roles in root development via auxin signaling mediated by genetic pathways. In this study, we found that the transcription factor ZmARF1 was associated with low inorganic phosphate (Pi) stress-related traits in maize. This superior root morphology and greater phosphate stress tolerance could be ascribed to the overexpression of ZmARF1. The knock out mutant zmarf1 had shorter primary roots, fewer root tip number, and lower root volume and surface area. Transcriptomic data indicate that ZmLBD1, a direct downstream target gene, is involved in lateral root development, which enhances phosphate starvation tolerance. A transcriptional activation assay revealed that ZmARF1 specifically binds to the GC-box motif in the promoter of ZmLBD1 and activates its expression. Moreover, ZmARF1 positively regulates the expression of ZmPHR1, ZmPHT1;2, and ZmPHO2, which are key transporters of Pi in maize. We propose that ZmARF1 promotes the transcription of ZmLBD1 to modulate lateral root development and Pi-starvation induced (PSI) genes to regulate phosphate mobilization and homeostasis under phosphorus starvation. In addition, ZmERF2 specifically binds to the ABRE motif of the promoter of ZmARF1 and represses its expression. Collectively, the findings of this study revealed that ZmARF1 is a pivotal factor that modulates root development and confers low-Pi stress tolerance through the transcriptional regulation of the biological function of ZmLBD1 and the expression of key Pi transport proteins.

Two all-biomass cellulose/amino acid spherical nanoadsorbents based on a tri-aldehyde spherical nanocellulose II amino acid premodification platform for the efficient removal of Cr(VI) and Cu(II).

Adsorbents consisting of spherical nanoparticles exhibit superior adsorption performance and hence, have immense potential for various applications. In this study, a tri-aldehyde spherical nanoadsorbent premodification platform (CTNAP), which can be grafted with various amino acids, was synthesized from corn stalk. Subsequently, two all-biomass spherical nanoadsorbents, namely, cellulose/l-lysine (CTNAP-Lys) and cellulose/L-cysteine (CTNAP-Cys), were prepared. The morphologies as well as chemical and crystal structures of the two adsorbents were studied in detail. Notably, the synthesized adsorbents exhibited two important characteristics, namely, a spherical nanoparticle morphology and cellulose II crystal structure, which significantly enhanced their adsorption performance. The mechanism of the adsorption of Cr(VI) onto CTNAP-Lys and that of Cu(II) onto CTNAP-Cys were studied in detail, and the adsorption capacities were determined to be as high as 361.69 (Cr(VI)) and 252.38 mg/g (Cu(II)). Using the proposed strategy, it should be possible to prepare other all-biomass cellulose/amino acid spherical nanomaterials with high functional group density for adsorption, medical, catalytic, analytical chemistry, corrosion, and photochromic applications.

Identification and Functional Analysis of Drought-Responsive Long Noncoding RNAs in Maize Roots.

Long noncoding RNAs (lncRNAs) are transcripts with lengths of more than 200 nt and limited protein-coding potential. They were found to play important roles in plant stress responses. In this study, the maize drought-tolerant inbred line AC7643 and drought-sensitive inbred line AC7729/TZSRW, as well as their recombinant inbred lines (RILs) were selected to identify drought-responsive lncRNAs in roots. Compared with non-responsive lncRNAs, drought-responsive lncRNAs had different sequence characteristics in length of genes and number of exons. The ratio of down-regulated lncRNAs induced by drought was significantly higher than that of coding genes; and lncRNAs were more widespread expressed in recombination sites in the RILs. Additionally, by integration of the modifications of DNA 5-methylcytidine (5mC), histones, and RNA N6-methyladenosine (m6A), it was found that the enrichment of histone modifications associated with transcriptional activation in the genes generated lncRNAs was lower that coding genes. The lncRNAs-mRNAs co-expression network, containing 15,340 coding genes and 953 lncRNAs, was constructed to investigate the molecular functions of lncRNAs. There are 13 modules found to be associated with survival rate under drought. We found nine SNPs located in lncRNAs among the modules associated with plant survival under drought. In conclusion, we revealed the characteristics of lncRNAs responding to drought in maize roots based on multiomics studies. These findings enrich our understanding of lncRNAs under drought and shed light on the complex regulatory networks that are orchestrated by the noncoding RNAs in response to drought stress.

TrichomeYOLO: A Neural Network for Automatic Maize Trichome Counting.

Plant trichomes are epidermal structures with a wide variety of functions in plant development and stress responses. Although the functional importance of trichomes has been realized, the tedious and time-consuming manual phenotyping process greatly limits the research progress of trichome gene cloning. Currently, there are no fully automated methods for identifying maize trichomes. We introduce TrichomeYOLO, an automated trichome counting and measuring method that uses a deep convolutional neural network, to identify the density and length of maize trichomes from scanning electron microscopy images. Our network achieved 92.1% identification accuracy on scanning electron microscopy micrographs of maize leaves, which is much better performed than the other 5 currently mainstream object detection models, Faster R-CNN, YOLOv3, YOLOv5, DETR, and Cascade R-CNN. We applied TrichomeYOLO to investigate trichome variations in a natural population of maize and achieved robust trichome identification. Our method and the pretrained model are open access in Github (https://github.com/yaober/trichomecounter). We believe TrichomeYOLO will help make efficient trichome identification and help facilitate researches on maize trichomes.

ZmLBD5, a class-II LBD gene, negatively regulates drought tolerance by impairing abscisic acid synthesis.

Lateral organ boundaries domain (LBD) proteins are plant-specific transcription factors. Class-I LBD genes have been widely demonstrated to play pivotal roles in organ development; however, knowledge on class-II genes remains limited. Here, we report that ZmLBD5, a class-II LBD gene, is involved in the regulation of maize (Zea mays) growth and the drought response by affecting gibberellin (GA) and abscisic acid (ABA) synthesis. ZmLBD5 is mainly involved in regulation of the TPS-KS-GA2ox gene module, which is comprised of key enzyme-encoding genes involved in GA and ABA biosynthesis. ABA insufficiency increases stomatal density and aperture in overexpression plants and causes a drought-sensitive phenotype by promoting water transpiration. Increased GA1 levels promotes seedling growth in overexpression plants. Accordingly, CRISPR/Cas9 knockout lbd5 seedlings are dwarf but drought-tolerant. Moreover, lbd5 has a higher grain yield under drought stress conditions and shows no penalty in well-watered conditions compared to the wild type. On the whole, ZmLBD5 is a negative regulator of maize drought tolerance, and it is a potentially useful target for drought resistance breeding.

Teosinte confers specific alleles and yield potential to maize improvement.

KEY MESSAGE: Teosinte improves maize grain yield and broadens the maize germplasm. Seventy-one quantitative trait loci associated with 24 differential traits between maize and teosinte were identified. Maize is a major cereal crop with a narrow germplasm that has limited its production and breeding progress. Teosinte, an ancestor of maize, provides valuable genetic resources for maize breeding. To identify the favorable alien alleles in teosinte and its yield potential for maize breeding, 4 backcrossed maize-teosinte recombinant inbred line (RIL) populations were cultivated under five conditions. A North Carolina mating design II experiment was conducted on inbred lines with B73 and Mo17 pedigree backgrounds to analyze their combining ability. Abundant phenotypic variation on 26 traits of four RIL populations were found, of which barren tip length, kernel height, and test weight showed positive genetic improvement potential. The hybrid FM132 (BD138/MP116) showed a superior grain yield to that of the check, with an average yield gain of 4.86%. Moreover, inbred lines BD138 and MP048 showed a higher general grain yield combining ability than those of their corresponding checks. We screened 4,964,439 high-quality single-nucleotide polymorphisms in the BD (B73/Zea diploperennis) RIL population for bin construction and used 2322 bin markers for genetic map construction and quantitative trait loci (QTL) mapping. Via inclusive composite interval mapping, 71 QTL associated with 24 differential traits were identified. Gene annotation and transcriptional expression suggested that Zm00001eb352570 and Zm00001eb352580, both annotated as ethylene-responsive transcription factors, were key candidate genes that regulate ear height and the ratio of ear to plant height. Our results indicate that teosinte could broaden the narrow maize germplasm, improve yield potential, and provide desirable alleles for maize breeding.

Identification of Fusarium verticillioides Resistance Alleles in Three Maize Populations With Teosinte Gene Introgression.

Fusarium ear rot (FER) is a common fungal disease in maize (Zea mays L.) caused by Fusarium verticillioides. Resistant germplasm resources for FER are rare in cultivated maize; however, teosintes (Z. mays ssp. parviglumis and Z. mays ssp. diploperennis), which are wild-type species of maize, have the potential to offer a novel source of resistance alleles to enhance pathogen resistance in modern maize. Therefore, the aim of this study was to identify favorable alleles that confer significant levels of resistance toward FER. Three populations of BC2F8 recombinant inbred lines (RILs) were developed by crossing two different teosintes, Z. diploperennis and Z. parviglumis, with maize inbred lines B73 and Zheng58, and were screened for FER resistance. We found that Z. diploperennis and Z. parviglumis had higher resistance toward F. verticillioides in the leaves than B73 and Zheng58. However, the resistance toward F. verticillioides in the leaf and ear was unrelated among RILs. FER resistance was positively correlated with grain yield in the B73 × diploperennis (BD) and Zheng58 × parviglumis (ZP) populations, partly because the quantitative trait loci (QTLs) of FER resistance and yield traits were located close together. Four coincident QTLs (qFERbd5.177, qFERbd10.140, qFERzp4.066, and qFERzp5.116) and two highly reliable resistance-yield synergistic QTLs (qFERbd10.140 and qFERzp4.066) were identified in the BD and ZP populations, opening up the possibility of breeding for FER resistance without reducing yield.

ABA-inducible DEEPER ROOTING 1 improves adaptation of maize to water deficiency.

Root architecture remodelling is critical for forage moisture in water-limited soil. DEEPER ROOTING 1 (DRO1) in Oryza, Arabidopsis, and Prunus has been reported to improve drought avoidance by promoting roots to grow downward and acquire water from deeper soil. In the present study, we found that ZmDRO1 responded more strongly to abscisic acid (ABA)/drought induction in Zea mays ssp. mexicana, an ancestral species of cultivated maize, than in B73. It was proposed that this is one of the reasons why Zea mays ssp. mexicana has a more noticeable change in the downward direction angle of the root and fewer biomass penalties under water-deficient conditions. Thus, a robust, synthetic ABA/drought-inducible promoter was used to control the expression of ZmDRO1B73 in Arabidopsis and cultivated maize for drought-resistant breeding. Interestingly, ABA-inducible ZmDRO1 promoted a larger downward root angle and improved grain yield by more than 40% under water-limited conditions. Collectively, these results revealed that different responses to ABA/drought induction of ZmDRO1 confer different drought avoidance abilities, and we demonstrated the application of ZmDRO1 via an ABA-inducible strategy to alter the root architecture of modern maize to improve drought adaptation in the field.

Characteristics of microRNAs and Target Genes in Maize Root under Drought Stress.

Maize (Zea mays) is an important multi-functional crop. The growth and yield of maize are severely affected by drought stress. Previous studies have shown that microRNAs (miRNAs) in maize play important roles in response to abiotic stress; however, their roles in response to drought stress in maize roots is unclear. In our study, we found 375 miRNAs in the roots of 16 inbred lines. Of the 16 lines, zma-MIR168, zma-MIR156, and zma-MIR166 were highly expressed, whereas zma-MIR399, zma-MIR2218, and zma-MIR2275 exhibited low expression levels. The expression patterns of miRNA in parental lines and their derived RILs are different. Over 50% of miRNAs exhibited a lower expression in recombinant inbred lines than in parents. The expression of 50 miRNAs was significantly altered under water stress (WS) in at least three inbred lines, and the expression of miRNAs in drought-tolerant lines changed markedly. To better understand the reasons for miRNA response to drought, the degree of histone modifications for miRNA genes was estimated. The methylation level of H3K4 and H3K9 in miRNA precursor regions changed more noticeably after WS, but no such phenomenon was seen for DNA methylation and m6A modification. After the prediction of miRNA targets using psRNATarget and psRobot, we used correlation analysis and qRT-PCR to further investigate the relationship between miRNAs and target genes. We found that 87 miRNA-target pairs were significantly negatively correlated. In addition, a weighted gene co-expression network analysis using miRNAs, as well as their predicted targets, was conducted to reveal that miR159, miR394, and miR319 may be related to maize root growth. The results demonstrated that miRNAs might play essential roles in the response to drought stress.

ZmLBD5 Increases Drought Sensitivity by Suppressing ROS Accumulation in Arabidopsis.

Drought stress is known to significantly limit crop growth and productivity. Lateral organ boundary domain (LBD) transcription factors-particularly class-I members-play essential roles in plant development and biotic stress. However, little information is available on class-II LBD genes related to abiotic stress in maize. Here, we cloned a maize class-II LBD transcription factor, ZmLBD5, and identified its function in drought stress. Transient expression, transactivation, and dimerization assays demonstrated that ZmLBD5 was localized in the nucleus, without transactivation, and could form a homodimer or heterodimer. Promoter analysis demonstrated that multiple drought-stress-related and ABA response cis-acting elements are present in the promoter region of ZmLBD5. Overexpression of ZmLBD5 in Arabidopsis promotes plant growth under normal conditions, and suppresses drought tolerance under drought conditions. Furthermore, the overexpression of ZmLBD5 increased the water loss rate, stomatal number, and stomatal apertures. DAB and NBT staining demonstrated that the reactive oxygen species (ROS) decreased in ZmLBD5-overexpressed Arabidopsis. A physiological index assay also revealed that SOD and POD activities in ZmLBD5-overexpressed Arabidopsis were higher than those in wild-type Arabidopsis. These results revealed the role of ZmLBD5 in drought stress by regulating ROS levels.

PPVED: A machine learning tool for predicting the effect of single amino acid substitution on protein function in plants.

Single amino acid substitution (SAAS) produces the most common variant of protein function change under physiological conditions. As the number of SAAS events in plants has increased exponentially, an effective prediction tool is required to help identify and distinguish functional SAASs from the whole genome as either potentially causal traits or as variants. Here, we constructed a plant SAAS database that stores 12 865 SAASs in 6172 proteins and developed a tool called Plant Protein Variation Effect Detector (PPVED) that predicts the effect of SAASs on protein function in plants. PPVED achieved an 87% predictive accuracy when applied to plant SAASs, an accuracy that was much higher than those from six human database software: SIFT, PROVEAN, PANTHER-PSEP, PhD-SNP, PolyPhen-2, and MutPred2. The predictive effect of six SAASs from three proteins in Arabidopsis and maize was validated with wet lab experiments, of which five substitution sites were accurately predicted. PPVED could facilitate the identification and characterization of genetic variants that explain observed phenotype variations in plants, contributing to solutions for challenges in functional genomics and systems biology. PPVED can be accessed under a CC-BY (4.0) license via http://www.ppved.org.cn.

Maize Transcription Factor ZmARF4 Confers Phosphorus Tolerance by Promoting Root Morphological Development.

Plant growth and development are closely related to phosphate (Pi) and auxin. However, data regarding auxin response factors (ARFs) and their response to phosphate in maize are limited. Here, we isolated ZmARF4 in maize and dissected its biological function response to Pi stress. Overexpression of ZmARF4 in Arabidopsis confers tolerance of Pi deficiency with better root morphology than wild-type. Overexpressed ZmARF4 can partially restore the absence of lateral roots in mutant arf7 arf19. The ZmARF4 overexpression promoted Pi remobilization and up-regulated AtRNS1, under Pi limitation while it down-regulated the expression of the anthocyanin biosynthesis genes AtDFR and AtANS. A continuous detection revealed higher activity of promoter in the Pi-tolerant maize P178 line than in the sensitive 9782 line under low-Pi conditions. Meanwhile, GUS activity was specifically detected in new leaves and the stele of roots in transgenic offspring. ZmARF4 was localized to the nucleus and cytoplasm of the mesophyll protoplast and interacted with ZmILL4 and ZmChc5, which mediate lateral root initiation and defense response, respectively. ZmARF4 overexpression also conferred salinity and osmotic stress tolerance in Arabidopsis. Overall, our findings suggest that ZmARF4, a pleiotropic gene, modulates multiple stress signaling pathways, and thus, could be a candidate gene for engineering plants with multiple stress adaptation.

Maize DNA Methylation in Response to Drought Stress Is Involved in Target Gene Expression and Alternative Splicing.

DNA methylation is important for plant growth, development, and stress response. To understand DNA methylation dynamics in maize roots under water stress (WS), we reanalyzed DNA methylation sequencing data to profile DNA methylation and the gene expression landscape of two inbred lines with different drought sensitivities, as well as two of their derived recombination inbred lines (RILs). Combined with genotyping-by-sequencing, we found that the inheritance pattern of DNA methylation between RILs and parental lines was sequence-dependent. Increased DNA methylation levels were observed under WS and the methylome of drought-tolerant inbred lines were much more stable than that of the drought-sensitive inbred lines. Distinctive differentially methylated genes were found among diverse genetic backgrounds, suggesting that inbred lines with different drought sensitivities may have responded to stress in varying ways. Gene body DNA methylation showed a negative correlation with gene expression but a positive correlation with exon splicing events. Furthermore, a positive correlation of a varying extent was observed between small interfering RNA (siRNA) and DNA methylation, which at different genic regions. The response of siRNAs under WS was consistent with the differential DNA methylation. Taken together, our data can be useful in deciphering the roles of DNA methylation in plant drought-tolerance variations and in emphasizing its function in alternative splicing.

A natural antisense transcript acts as a negative regulator for the maize drought stress response gene ZmNAC48.

Although plant-specific NAC transcription factors play crucial roles in response to abiotic stress, few reports describe the regulation of NAC genes in maize (Zea mays) by the cis-natural antisense transcripts (cis-NATs). In this study, 521 NAC genes from Gramineae were classified, of which 51 NAC genes contained cis-NATs. ZmNAC48 and cis-NATZmNAC48 co-localized to the same cell nucleus, and both transcripts responded to drought stress. Arabidopsis plants overexpressing ZmNAC48 had improved drought tolerance, lower rate of water loss, enhanced stomatal closure, and higher rates of survival. Transient expression in both maize protoplasts and tobacco leaves indicated that cis-NATZmNAC48 reduced ZmNAC48 expression. Western blotting and ribosome profiling analyses confirmed that cis-NATZmNAC48 lacked protein coding potential. Furthermore, the cis-NAT-derived small-interfering RNAs (nat-siRNAs) generated from the overlapping regions of ZmNAC48 and cis-NATZmNAC48 were detected in maize and transgenic Arabidopsis. Cis-NATZmNAC48 overexpressing maize showed higher water loss rate, increased stomatal opening, and had more dead leaves. Expression of ZmNAC48 and nat-siRNA was decreased in these plants. Taken together, our study indicates that both ZmNAC48 and cis-NATZmNAC48 are involved in plant drought stress responses, and that the double-stranded RNA-dependent mechanism is involved in the interaction between cis-NATZmNAC48 and ZmNAC48. Additionally, cis-NATZmNAC48 may negatively regulate ZmNAC48 to affect stomatal closure of maize.

Nutritional and physicochemical characteristics of purple sweet corn juice before and after boiling.

Sweet corn juice is becoming increasingly popular in China. In order to provide valuable health-related information to consumers, the nutritional and physicochemical characteristics of raw and boiled purple sweet corn juices were herein investigated. Sugars, antinutrients, total free phenols, anthocyanins, and antioxidant activity were analyzed by conventional chemical methods. The viscosity and stability of juices were determined by Ubbelohde viscosity meter and centrifugation, respectively. Boiling process could elevate viscosity, stability and sugar content, and reduce antinutrients, total free phenols, anthocyanins, and antioxidant activity in corn juice. In addition, short time boiling efficiently reduced the degradation of anthocyanins during subsequent refrigeration. The content of amino acids, vitamin B1/B2 and E were detected by High Performance Liquid Chromatography. Gas Chromatography Mass Spectrometry was used for the analysis of fatty acids and aroma compounds. Several aroma compounds not previously reported in corn were identified, including 1-heptanol, 2-methyl-2-butenal, (Z)-3-nonen-1-ol, 3-ethyl-2-methyl-1,3-hexadiene, and 2,4-bis(1,1-dimethylethyl)phenol. Interestingly, the boiling process had no apparent effect on the amino acids profile, but it caused a 45.8% loss of fatty acids in the juice by promoting the retention of fatty acids in the corn residue. These results provide detailed information that could be used for increasing consumers' knowledge of sweet corn juice, further development of sweet corn juice by food producers, and maize breeding programs.

Lateral mechanical impedance rather than frontal promotes cortical expansion of roots.

It has long been considered that mechanical impedance on root will restrict root elongation and consequently promote radial growth. When seedlings grew in sands, we did observe radial expansion of roots and it, however, arose before elongation restriction. Mechanical impedance of sands can be classified into frontal- and lateral-type based on the interaction site of root. Therefore, we suspected that radial expansion might be mainly stimulated by lateral- rather than frontal-impedance. To verify our speculation, roots were exposed to frontal- and lateral-impedance separately. Small plastic caps were used to provide pure frontal impedance on root tips and cylindrical plastic containers were used to provide pure lateral impedance. Root elongation was remarkably suppressed under the frontal impedance of plastic caps, and more than that in sand-condition. However, the radial expansion of the plastic-cap-fitted roots was far inferior to that of the sand-cultured roots. Microstructural analysis revealed that sand-condition thickened root largely dependents on cortical expansion, whereas plastic cap did it mainly by thickening stele. In cylindrical plastic containers, mechanical impedance came only from the lateral direction and promoted the expansion of cortex like sand-condition. Thus, we proposed that the expansion of cortex and the consequent radial growth of roots were mainly due to lateral impedance when seedlings grew in sands.

IDD16 negatively regulates stomatal initiation via trans-repression of SPCH in Arabidopsis.

In Arabidopsis, the initiation and proliferation of stomatal lineage cells is controlled by SPEECHLESS (SPCH). Phosphorylation of SPCH at the post-translational level has been reported to regulate stomatal development. Here we report that IDD16 acts as a negative regulator for stomatal initiation by directly regulating SPCH transcription. In Arabidopsis, IDD16 overexpression decreased abaxial stomatal density in a dose-dependent manner. Time course analysis revealed that the initiation of stomatal precursor cells in the IDD16-OE plants was severely inhibited. Consistent with these findings, the transcription of SPCH was greatly repressed in the IDD16-OE plants. In contrast, IDD16-RNAi transgenic line resulted in enhanced stomatal density, suggesting that IDD16 is an intrinsic regulator of stomatal development. ChIP analysis indicated that IDD16 could directly bind to the SPCH promoter. Furthermore, Arabidopsis plants overexpressing IDD16 exhibited significantly increased drought tolerance and higher integrated water use efficiency (WUE) due to reduction in leaf transpiration. Collectively, our results established that IDD16 negatively regulates stomatal initiation via trans-repression of SPCH, and thus provide a practical tool for increasing plant WUE through the manipulation of IDD16 expression.