Taxonomic identification and antagonistic activity of Streptomyces luomodiensis sp. nov. against phytopathogenic fungi.

Banana wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) is a devastating fungal disease. Biocontrol strategies hold immense potential for inhibiting the spread of Foc TR4. Here, 30 actinobacteria were isolated from soils and screened for their antagonistic activity against Foc TR4. Strain SCA4-21T was selected due to its strongest antagonistic activity against Foc TR4. Strain SCA4-21T also exhibited strong antagonistic activity against the other eight phytopathogenic fungi. The strain was identified as the genus Streptomyces according to its physiological, biochemical, and phenotypic characteristics. The phylogenetic trees of 16S rRNA sequences demonstrated that strain SCA4-21T formed a subclade with S. iranensis HM 35T and/or S. rapamycinicus NRRL B-5491T with low bootstrap values. Considering that 16S rRNAs did not provide sufficient resolution for species-level identification, the whole genome of strain SCA4-21T was sequenced. Multilocus sequence analysis (MLSA) based on five housekeeping gene alleles (atpD, gyrB, recA, rpoB, and trpB) revealed that strain SCA4-21T clustered into S. hygroscopicus subsp. hygroscopicus NBRC 13472T with 100% of bootstrap value. The analysis of the genome-based phylogeny also approved the results. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were 91.26 and 44.30%, respectively, with values below the respective species level threshold of 95 and 70%. Hence, strain SCA 4-21T represented a novel species within the genus Streptomyces, named Streptomyces luomodiensis sp. nov. The type strain is SCA4-21T (=GDMCC4.340T = JCM36555T). By the CAZymes analysis, 348 carbohydrate-active enzymes (CAZymes) were detected, including 15 chitinases and eight ß-1,3-glucanases. The fermentation broth of strain SCA4-21T, exhibiting strong antagonistic activity against Foc TR4, demonstrated high activities of chitinase and ß-1,3-glucanase, which might be involved in antifungal activity. Our results showed an innovative potential biocontrol agent for managing plant fungal diseases, specifically banana fusarium wilt.

TIP aquaporins in Cyperus esculentus: genome-wide identification, expression profiles, subcellular localizations, and interaction patterns.

BACKGROUND: Tonoplast intrinsic proteins (TIPs), which typically mediate water transport across vacuolar membranes, play an essential role in plant growth, development, and stress responses. However, their characterization in tigernut (Cyperus esculentus L.), an oil-bearing tuber plant of the Cyperaceae family, is still in the infancy. RESULTS: In this study, a first genome-wide characterization of the TIP subfamily was conducted in tigernut, resulting in ten members representing five previously defined phylogenetic groups, i.e., TIP1-5. Although the gene amounts are equal to that present in two model plants Arabidopsis and rice, the group composition and/or evolution pattern were shown to be different. Except for CeTIP1;3 that has no counterpart in both Arabidopsis and rice, complex orthologous relationships of 1:1, 1:2, 1:3, 2:1, and 2:2 were observed. Expansion of the CeTIP subfamily was contributed by whole-genome duplication (WGD), transposed, and dispersed duplications. In contrast to the recent WGD-derivation of CeTIP3;1/-3;2, synteny analyses indicated that TIP4 and - 5 are old WGD repeats of TIP2, appearing sometime before monocot-eudicot divergence. Expression analysis revealed that CeTIP genes exhibit diverse expression profiles and are subjected to developmental and diurnal fluctuation regulation. Moreover, when transiently overexpressed in tobacco leaves, CeTIP1;1 was shown to locate in the vacuolar membrane and function in homo/heteromultimer, whereas CeTIP2;1 is located in the cell membrane and only function in heteromultimer. Interestingly, CeTIP1;1 could mediate the tonoplast-localization of CeTIP2;1 via protein interaction, implying complex regulatory patterns. CONCLUSIONS: Our findings provide a global view of CeTIP genes, which provide valuable information for further functional analysis and genetic improvement through manipulating key members in tigernut.

Biological function research of Fusarium oxysporum f. sp. cubense inducible banana long noncoding RNA Malnc2310 in Arabidopsis.

Long noncoding RNAs (lncRNAs) participate in plant biological processes under biotic and abiotic stresses. However, little is known about the function and regulation mechanism of lncRNAs related to the pathogen at a molecular level. A banana lncRNA, Malnc2310, is a Fusarium oxysporum f. sp. cubense inducible lncRNA in roots. In this study, we demonstrate the nuclear localization of Malnc2310 by fluorescence in situ hybridization and it can bind to several proteins that are related to flavonoid pathway, pathogen response and programmed cell death. Overexpression of Malnc2310 increases susceptibility to Fusarium crude extract (Fu), salinity, and cold in transgenic Arabidopsis. In addition, Malnc2310 transgenic Arabidopsis accumulated more anthocyanins under Fusarium crude extract and cold treatments that are related to upregulation of these genes involved in anthocyanin biosynthesis. Based on our findings, we propose that Malnc2310 may participate in flavonoid metabolism in plants under stress. Furthermore, phenylalanine ammonia lyase (PAL) protein expression was enhanced in Malnc2310 overexpressed transgenic Arabidopsis, and Malnc2310 may participate in PAL regulation by binding to it. This study provides new insights into the role of Malnc2310 in mediating plant stress adaptation.

The cassava (Manihot-esculenta Crantz)'s nitrate transporter NPF4.5, expressed in seedling roots, involved in nitrate flux and osmotic stress.

AtNPF4.5/AIT2, which was predicted to be a low-affinity transporter capable for nitrate uptake, was screened by ABA receptor complex in Arabidopsis ten years ago. However, the molecular and biochemical characterizations of AtNPF4.5 in plants remained largely unclear. In this study, the function of a plasma-membrane-localized and root-specifically-expressed gene MeNPF4.5 (Manihot-esculenta NITRATE TRANSPORTER 1 PTR FAMILY4.5), an ortholog of the Arabidopsis thaliana NPF4.5, was investigated in cassava roots as a nitrate efflux transporter on low nitrate medium and an influx transporter following exposure to high concentration of external nitrates. Moreover, RNA interference (RNAi) of MeNPF4.5 reduced the nitrate efflux capacity but the overexpressing cassava seedlings increased the ability of efflux from the elongation to the mature zone of root under low nitrate treatments. Besides, MeNPF4.5-RNAi expression reduced the nitrate influx capacity but enhanced nitrate absorption in parts of overexpressing plants from the meristem, elongation to mature zone of roots under high nitrate conditions. Furthermore, MeNPF4.5-RNAi seedlings survived owing to roots that could grow normally, but the MeNPF4.5-over-expressors showed adverse growth under 7% PEG6000 stress, suggesting that MeNPF4.5 negatively regulated the osmotic stress and was involved in nitrate flux through cassava seedlings.

CC-type glutaredoxin, MeGRXC3, associates with catalases and negatively regulates drought tolerance in cassava (Manihot esculenta Crantz).

Glutaredoxins (GRXs) are essential for reactive oxygen species (ROS) homeostasis in responses of plants to environment changes. We previously identified several drought-responsive CC-type GRXs in cassava, an important tropical crop. However, how CC-type GRX regulates ROS homeostasis of cassava under drought stress remained largely unknown. Here, we report that a drought-responsive CC-type GRX, namely MeGRXC3, was associated with activity of catalase in the leaves of 100 cultivars (or unique unnamed genotypes) of cassava under drought stress. MeGRXC3 negatively regulated drought tolerance by modulating drought- and abscisic acid-induced stomatal closure in transgenic cassava. It antagonistically regulated hydrogen peroxide (H2 O2 ) accumulation in epidermal cells and guard cells. Moreover, MeGRXC3 interacted with two catalases of cassava, MeCAT1 and MeCAT2, and regulated their activity in vivo. Additionally, MeGRXC3 interacts with a cassava TGA transcription factor, MeTGA2, in the nucleus, and regulates the expression of MeCAT7 through a MeTGA2-MeMYB63 pathway. Overall, we demonstrated the roles of MeGRXC3 in regulating activity of catalase at both transcriptional and post-translational levels, therefore involving in ROS homeostasis and stomatal movement in responses of cassava to drought stress. Our study provides the first insights into how MeGRXC3 may be used in molecular breeding of cassava crops.

Cassava (Manihot esculenta) Slow Anion Channel (MeSLAH4) Gene Overexpression Enhances Nitrogen Assimilation, Growth, and Yield in Rice.

Nitrogen is one of the most important nutrient elements required for plant growth and development, which is also immensely related to the efficient use of nitrogen by crop plants. Therefore, plants evolved sophisticated mechanisms and anion channels to extract inorganic nitrogen (nitrate) from the soil or nutrient solutions, assimilate, and recycle the organic nitrogen. Hence, developing crop plants with a greater capability of using nitrogen efficiently is the fundamental research objective for attaining better agricultural productivity and environmental sustainability. In this context, an in-depth investigation has been conducted into the cassava slow type anion channels (SLAHs) gene family, including genome-wide expression analysis, phylogenetic relationships with other related organisms, chromosome localization, and functional analysis. A potential and nitrogen-responsive gene of cassava (MeSLAH4) was identified and selected for overexpression (OE) analysis in rice, which increased the grain yield and root growth related performance. The morpho-physiological response of OE lines was better under low nitrogen (0.01 mm NH4NO3) conditions compared to the wild type (WT) and OE lines under normal nitrogen (0.5 mm NH4NO3) conditions. The relative expression of the MeSLAH4 gene was higher (about 80-fold) in the OE line than in the wild type. The accumulation and flux assay showed higher accumulation of NO 3 - and more expansion of root cells and grain dimension of OE lines compared to the wild type plants. The results of this experiment demonstrated that the MeSLAH4 gene may play a vital role in enhancing the efficient use of nitrogen in rice, which could be utilized for high-yielding crop production.

Natural variation MeMYB108 associated with tolerance to stress-induced leaf abscission linked to enhanced protection against reactive oxygen species in cassava.

KEY MESSAGE: Natural variation of the MeMYB108 exon was associated with reactive oxygen scavengers led to alleviate leaf abscission under drought in cassava. The reactive oxygen scavengers play important roles in regulating the cassava (Manihot esculenta Crantz) leaf abscission induced by stresses. To date, the relationship between natural variations of MYB genes and reactive oxygen scavengers under drought in cassava genotypes remains unclear. Here, we reported the transcription factor MeMYB108 played an important role in regulating leaf abscission exposed to drought in cassava. The expression levels of MeMYB108 in abscission zones of cassava leaf pulvinus were higher in cassava genotype SC124, which were less easy to shed leaves under stress than cassava genotype SC8 when the leaf abscission induced by the same drought condition. Compared with wild type and interference expression plants, overexpression of MeMYB108 significantly reduced the drought-induced leaf abscission rate under drought. The consecutively 2-year analysis of reactive oxygen scavengers showed significant differences among different cassava genotypes under drought-induced leaf abscission, indicating the relevance between reactive oxygen scavengers and leaf abscission. Correlation analysis revealed the natural variation of the MeMYB108 exon was associated with reactive oxygen scavengers during drought-induced leaf abscission. Association analysis between pairwise LD of DNA polymorphism indicated the MeMYB108 allele enhanced the tolerance of cassava to drought-induced leaf abscission. Complementation transgenic lines containing the elite allele of MeMYB108 SC124 decreased the leaf abscission rate induced by drought conditions, demonstrating natural variation in MeMYB108 contributed to leaf abscission tolerance induced by drought in cassava. Further studies showed MeMYB108 played an active role in the tolerance of cassava to drought-induced leaf abscission by inducing scavenging of reactive oxygen species.

Biocontrol potential and antifungal mechanism of a novel Streptomyces sichuanensis against Fusarium oxysporum f. sp. cubense tropical race 4 in vitro and in vivo.

Most commercial banana cultivars are highly susceptible to Fusarium wilt caused by soilborne fungus Fusarium oxysporum f. sp. cubense (Foc), especially tropical race 4 (TR4). Biological control using antagonistic microorganism has been considered as an alternative method to fungicide. Our previous study showed that Streptomyces sp. SCA3-4 T had a broad-spectrum antifungal activity from the rhizosphere soil of Opuntia stricta in a dry hot valley. Here, the sequenced genome of strain SCA3-4 T contained 6614 predicted genes with 72.38% of G + C content. A polymorphic tree was constructed using the multilocus sequence analysis (MLSA) of five house-keeping gene alleles (atpD, gyrB, recA, rpoB, and trpB). Strain SCA3-4 T formed a distinct clade with Streptomyces mobaraensis NBRC 13819 T with 71% of bootstrap. Average nucleotide identity (ANI) values between genomes of strain SCA3-4 T and S. mobaraensis NBRC 13819 T was 85.83% below 95-96% of the novel species threshold, and named after Streptomyces sichuanensis sp. nov. The type strain is SCA3-4 T (= GDMCC 4.214 T = JCM 34964 T). Genomic analysis revealed that strain SCA3-4 T contained 36 known biosynthetic gene clusters of secondary metabolites. Antifungal activity of strain SCA3-4 T was closely associated with the production of siderophore and its extracts induced the apoptosis of Foc TR4 cells. A total of 12 potential antifungal metabolites including terpenoids, esters, acid, macrolides etc. were obtained by the gas chromatography-mass spectrometry (GC-MS). Greenhouse experiment indicated that strain SCA3-4 T could significantly inhibit infection of Foc TR4 in the roots and corms of banana seedlings and reduce disease index. Therefore, strain SCA3-4 T is an important microbial resource for exploring novel natural compounds and developing biopesticides to manage Foc TR4. KEY POINTS: • Strain SCA3-4 T was identified as a novel species of Streptomyces. • Siderophore participates in the antifungal regulation. • Secondary metabolites of strain SCA3-4 T improves the plant resistance to Foc TR4.

Global identification of full-length cassava lncRNAs unveils the role of cold-responsive intergenic lncRNA 1 in cold stress response.

Long noncoding RNAs (lncRNAs) have been considered to be important regulators of gene expression in a range of biological processes in plants. A large number of lncRNAs have been identified in plants. However, most of their biological functions still remain to be determined. Here, we identified a total of 3004 lncRNAs in cassava under normal or cold-treated conditions from Iso-seq data. We further characterized a cold-responsive intergenic lncRNA 1 (CRIR1) as a novel positive regulator of the plant response to cold stress. CRIR1 can be significantly induced by cold treatment. Ectopic expression of CRIR1 in cassava enhanced the cold tolerance of transgenic plants. Transcriptome analysis demonstrated that CRIR1 regulated a range of cold stress-related genes in a CBF-independent pathway. We further found that CRIR1 RNA can interact with cassava cold shock protein 5 (MeCSP5), which acts as an RNA chaperone, indicating that CRIR1 may recruit MeCSP5 to improve the translation efficiency of messenger RNA. In summary, our study extends the repertoire of lncRNAs in plants as well as their role in cold stress responses. Moreover, it reveals a mechanism by which CRIR1 affected cold stress response by modulating the expression of stress-responsive genes and increasing their translational yield.

Identification and Antifungal Mechanism of a Novel Actinobacterium Streptomyces huiliensis sp. nov. Against Fusarium oxysporum f. sp. cubense Tropical Race 4 of Banana.

Banana is an important fruit crop. Fusarium wilt caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) seriously threatens the global banana industry. It is difficult to control the disease spread using chemical measures. In addition, commercial resistant cultivars are also lacking. Biological control is considered as a promising strategy using antagonistic microbes. Actinomycetes, especially Streptomyces, are potential sources of producing novel bioactive secondary metabolites. Here, strain SCA2-4 T with strong antifungal activity against Foc TR4 was isolated from the rhizospheric soil of Opuntia stricta in a dry hot valley. The morphological, physiological and chemotaxonomic characteristics of the strain were consistent with the genus Streptomyces. Based on the homology alignment and phylogenetic trees of 16S rRNA gene, the taxonomic status of strain SCA2-4 T exhibited a paradoxical result and low bootstrap value using different algorithms in the MEGA software. It prompted us to further discriminate this strain from the closely related species by the multilocus sequence analysis (MLSA) using five house-keeping gene alleles (atpD, gyrB, recA, rpoB, and trpB). The MLSA trees calculated by three algorithms demonstrated that strain SCA2-4 T formed a distinct clade with Streptomyces mobaraensis NBRC 13819 T . The MLSA distance was above 0.007 of the species cut-off. Average nucleotide identity (ANI) values between strain SCA2-4 T genome and two standard strain genomes were below 95-96% of the novel species threshold. Strain SCA2-4 T was assigned to a novel species of the genus Streptomyces and named as Streptomyces huiliensis sp. nov. The sequenced complete genome of SCA2-4 T encoded 51 putative biosynthetic gene clusters of secondary metabolites. Genome alignment revealed that ten gene clusters were involved in the biosynthesis of antimicrobial metabolites. It was supported that strain SCA2-4 T showed strong antifungal activities against the pathogens of banana fungal diseases. Extracts abstracted from the culture filtrate of strain SCA2-4 T seriously destroyed cell structure of Foc TR4 and inhibited mycelial growth and spore germination. These results implied that strain SCA2-4 T could be a promising candidate for biological control of banana Fusarium wilt.

Expression of the cassava nitrate transporter NRT2.1 enables Arabidopsis low nitrate tolerance.

The cassava grows well on low-nutrient soils because of its high-affinity to absorb nitrate. However, the molecular mechanisms by which cassava adapts itself to this environment remain elusive, although we have cloned a putative gene named MeNRT2.1 which has a crucial role in high-affinity nitrate transporter from cassava seeding. Here, the expression pattern of MeNRT2.1 was further assessed using the GUS activity driven by MeNRT2.1 promoter in Arabidopsis transformation plants. The GUS activity was monitored over time following the reduction of nitrate supply. The GUS gene expression not only peaked in roots after 12 h in 0.2mM nitrate media, but also stained stems and leaves. Arabidopsis plants with overexpression of MeNRT2.1 increased the biomass compared to the wild type on rich nitrogen (N-full) media. However, chlorate sensitivity analysis showed that Arabidopsis plants expressing MeNRT2.1 were more susceptable to chlorate than wild type. Significantly, after growing for 15 days on media containing 0.2mM nitrate concentration, wild-type plants became yellowor died, while the transgenic MeNRT2.1 Arabidopsis plants maintained normal growth. With significant increases in the amount of 15NO- 3 uptake in roots, the MeNRT2.1 plants also increased the contents of chlorophyll and nitrate reductase. Taken together, these results demonstrate that MeNRT2.1 has an important role in adaptation to low nitrate concentration as a nitrate transporter.

Taxonomy and Broad-Spectrum Antifungal Activity of Streptomyces sp. SCA3-4 Isolated From Rhizosphere Soil of Opuntia stricta.

Actinobacteria are important producers of bioactive compounds. Extreme ecosystems cause evolution of novel secondary metabolic pathways of Actinobacteria and increase the possible discovery of new biological functions of bioactive compounds. Here, we isolated 65 Actinobacteria from rhizosphere soil samples of Opuntia stricta. An Actinobacteria strain (named SCA3-4) was screened against Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4, ATCC 76255). The strain produced pink-white aerial mycelia and brown substrate mycelium on Gause No. 1 agar. Biverticillate chains of cylindrical spores were observed by scanning electron microscopy (SEM). Based on alignment of 16S rRNA sequences, a constructed phylogenetic tree showed that strain SCA3-4 shared a 99.54% similarity with Streptomyces lilacinus NRRL B-1968T. The morphological, biochemical, physiological, and molecular characteristics further indicated that strain SCA3-4 belongs to the Streptomyces sp. It can grow well on medium with the following antibiotics chloramphenicol, streptomycin, penicillin-G, gentamicin, erythromycin, nystatin or neomycin sulfate. The polymerase chain reaction (PCR) amplification of types I and II polyketide synthase genes (PKS-I and PKS-II) suggested its bioactive potential. Under treatment with 100 µg/ml of ethyl acetate extracts isolated from Streptomyces sp. SCA3-4, growth of Foc TR4 was inhibited and cell membrane was destroyed. Crude extracts also showed a broad-spectrum antifungal activity against 13 phytopathogenic fungi including Foc TR4 and displayed the lowest minimum inhibitory concentration (MIC) (0.781 µg/ml) against Colletotrichum fragariae (ATCC 58718). A total of 21 different compounds identified by gas chromatography-mass spectrometry (GC-MS) were composed of phenolic compound, pyrrolizidine, hydrocarbons, esters, and acids. Besides the known active compounds, Streptomyces sp. SCA3-4 possesses antimicrobial or other biological activities. Further attention will be paid on other compounds with no functional annotation, aiming at the discovery of new bioactive substances.

Phylogeny and expression pattern analysis of TCP transcription factors in cassava seedlings exposed to cold and/or drought stress.

The TCP transcription factors usually act as integrators of multiple growth regulatory and environmental stimuli. However, little is known about this gene family in the important tropical crop cassava (Manihot esculenta). In this study, 36 TCP genes were identified and renamed based on cassava whole-genome sequence and their sequence similarity with Arabidopsis TCPs. Typical TCP domains were detected in these proteins by multiple sequence alignment analysis. Evolutionary analysis indicated that MeTCPs could be divided into 8 subgroups, which was further supported by gene structure and conserved motif analyses. qRT-PCR analysis revealed tissue-specific and hormone-responsive expression patterns of MeTCP genes. Moreover, with global expression and promoter analysis, we found that MeTCPs showed similar or distinct expression patterns under cold and/or drought stress, suggesting that they might participate in distinct signaling pathways. Our study provides the first comprehensive analysis of TCP gene family in the cassava genome. The data will be useful for uncovering the potential functions of MeTCP genes, and their possible roles in mediating hormone and abiotic stress responses in cassava.

Molecular diversity analysis, drought related marker-traits association mapping and discovery of excellent alleles for 100-day old plants by EST-SSRs in cassava germplasms (Manihot esculenta Cranz).

Cassava is the third largest food crop of the world and has strong ability of drought tolerance. In order to evaluate the molecular diversity and to discover novel alleles for drought tolerance in cassava germplasms, we examined a total of 107 abiotic stress related expressed sequence tags-simple sequence repeat (EST-SSR) markers in 134 cassava genotypes coming from planting regions worldwide and performed drought related marker-traits association mapping. As results, we successfully amplified 98 of 107 markers in 97 polymorphic loci and 279 alleles, with 2.87 alleles per locus, gene diversity of 0.48 and polymorphic information content (PIC) of 0.41 on average. The genetic coefficient between every two lines was 0.37 on average, ranging from 0.21 to 0.82. According to our population structure analysis, these samples could be divided into three sub-populations showing obvious gene flow between them. We also performed water stress experiments using 100-day old cassava plants in two years and calculated the drought tolerance coefficients (DTCs) and used them as phenotypes for marker-trait association mapping. We found that 53 markers were significantly associated with these drought-related traits, with a contribution rate for trait variation of 8.60% on average, ranging between 2.66 and 28.09%. Twenty-four of these 53 associated genes showed differential transcription or protein levels which were confirmed by qRT-PCR under drought stress when compared to the control conditions in cassava. Twelve of twenty-four genes were the same differential expression patterns in omics data and results of qRT-PCR. Out of 33 marker-traits combinations on 24 loci, 34 were positive and 53 negative alleles according to their phenotypic effects and we also obtained the typical materials which carried these elite alleles. We also found 23 positive average allele effects while 10 loci were negative according to their allele effects (AAEs). Our results on molecular diversity, locus association and differential expression under drought can prove beneficial to select excellent materials through marker assisted selection and for functional genes research in the future.

Leaf rolling controlled by the homeodomain leucine zipper class IV gene Roc5 in rice.

Leaf rolling is considered an important agronomic trait in rice (Oryza sativa) breeding. To understand the molecular mechanism controlling leaf rolling, we screened a rice T-DNA insertion population and isolated the outcurved leaf1 (oul1) mutant showing abaxial leaf rolling. The phenotypes were caused by knockout of Rice outermost cell-specific gene5 (Roc5), an ortholog of the Arabidopsis (Arabidopsis thaliana) homeodomain leucine zipper class IV gene GLABRA2. Interestingly, overexpression of Roc5 led to adaxially rolled leaves, whereas cosuppression of Roc5 resulted in abaxial leaf rolling. Bulliform cell number and size increased in oul1 and Roc5 cosuppression plants but were reduced in Roc5-overexpressing lines. The data indicate that Roc5 negatively regulates bulliform cell fate and development. Gene expression profiling, quantitative polymerase chain reaction, and RNA interference (RNAi) analyses revealed that Protodermal Factor Like (PFL) was probably down-regulated in oul1. The mRNA level of PFL was increased in Roc5-overexpressing lines, and PFL-RNAi transgenic plants exhibit reversely rolling leaves by reason of increases of bulliform cell number and size, indicating that Roc5 may have a conserved function. These are, to our knowledge, the first functional data for a gene encoding a homeodomain leucine zipper class IV transcriptional factor in rice that modulates leaf rolling.