WEG1, which encodes a cell wall hydroxyproline-rich glycoprotein,is essential for parental root elongation controlling lateral root formation in rice

Lateral roots (LRs) determine the overall root system architecture, thus enabling plants to efficiently explore their underground environment for water and nutrients. However, the mechanisms regulating LR development are poorly understood in monocotyledonous plants. We characterized a rice mutant, wavy root elongation growth 1 (weg1), that produced higher number of long and thick LRs (L-type LRs) formed from the curvatures of its wavy parental roots caused by asymmetric cell growth in the elongation zone. Consistent with this phenotype, was the expression of the WEG1 gene, which encodes a putative member of the hydroxyproline-rich glycoprotein family that regulates cell wall extensibility, in the root elongation zone. The asymmetric elongation growth in roots is well known to be regulated by auxin, but we found that the distribution of auxin at the apical region of the mutant and the wild-type roots was symmetric suggesting that the wavy root phenotype in rice is independent of auxin. However, the accumulation of auxin at the convex side of the curvatures, the site of L-type LR formation, suggested that auxin likely induced the formation of L-type LRs. This was supported by the need of a high amount of exogenous auxin to induce the formation of L-type LRs. These results suggest that the MNU-induced weg1 mutated gene regulates the auxin-independent parental root elongation that controls the number of likely auxin-induced L-type LRs, thus reflecting its importance in improving rice root architecture.     

Cadmium decreases crown root number by decreasing endogenous nitric oxide,which is indispensable for crown root primordia initiation in rice seedlings

Cadmium (Cd) is toxic to crown roots (CR), which are essential for maintaining normal growth and development in rice seedlings. Nitric oxide (NO) is an important signaling molecule that plays a pivotal role in plant root organogenesis. Here, the effects of Cd on endogenous NO content and root growth conditions were studied in rice seedlings. Results showed that similar to the NO scavenger, cPTIO, Cd significantly decreased endogenous NO content and CR number in rice seedlings, and these decreases were recoverable with the application of sodium nitroprusside (SNP, a NO donor). Microscopic analysis of root collars revealed that treatment with Cd and cPTIO inhibited CR primordia initiation. In contrast, although SNP partially recovered Cd-caused inhibition of CR elongation, treatment with cPTIO had no effect on CR elongation. l-NMMA, a widely used nitric oxide synthase (NOS) inhibitor, decreased endogenous NO content and CR number significantly, while tungstate, a nitrate reductase (NR) inhibitor, had no effect on endogenous NO content and CR number. Moreover, enzyme activity assays indicated that treatment with SNP inhibited NOS activity significantly, but had no effect on NR activity. All these results support the conclusions that a critical endogenous NO concentration is indispensable for rice CR primordia initiation rather than elongation, NOS is the main source for endogenous NO generation, and Cd decreases CR number by inhibiting NOS activity and thus decreasing endogenous NO content in rice seedlings.     

OsPIN2, which encodes a member of the auxin efflux carrier proteins,is involved in root elongation growth and lateral root formation patterns via the regulation of auxin distribution in rice

Auxin flow is important for different root developmental processes such as root formation, emergence, elongation and gravitropism. However, the detailed information about the mechanisms regulating the auxin flow is less well understood in rice. We characterized the auxin transport‐related mutants, Ospin‐formed2‐1 (Ospin2‐1) and Ospin2‐2, which exhibited curly root phenotypes and altered lateral root formation patterns in rice. The OsPIN2 gene encodes a member of the auxin efflux carrier proteins that possibly regulates the basipetal auxin flow from the root tip toward the root elongation zone. According to DR5‐driven GUS expression, there is an asymmetric auxin distribution in the mutants that corresponded with the asymmetric cell elongation pattern in the mutant root tip. Auxin transport inhibitor, N‐1‐naphthylphthalamic acid and Ospin2‐1 Osiaa13 double mutant rescued the curly root phenotype indicating that this phenotype results from a defect in proper auxin distribution. The typical curly root phenotype was not observed when Ospin2‐1 was grown in distilled water as an alternative to tap water, although higher auxin levels were found at the root tip region of the mutant than that of the wild‐type. Therefore, the lateral root formation zone in the mutant was shifted basipetally compared with the wild‐type. These results reflect that an altered auxin flow in the root tip region is responsible for root elongation growth and lateral root formation patterns in rice.     

The effects of auxin on lateral root initiation and root gravitropism in a lateral rootless mutant Lrt1 of rice (Oryza sativa L.)

Auxins control growth and development in plants, including lateral rootinitiation and root gravity response. However, how endogenous auxin regulatesthese processes is poorly understood. In this study, the effects of auxins onlateral root initiation and root gravity response in rice were investigatedusing a lateral rootless mutant Lrt1, which fails to formlateral roots and shows a reduced root gravity response. Exogenous applicationof IBA to the Lrt1 mutant restored both lateral rootinitiation and root gravitropism. However, application of IAA, a major form ofnatural auxin, restored only root gravitropic response but not lateral rootinitiation. These results suggest that IBA is more effective than IAA in lateralroot formation and that IBA also plays an important role in root gravitropicresponse in rice. The application of NAA restored lateral root initiation, butdid not completely restore root gravitropism. Root elongation assays ofLrt1 displayed resistance to 2,4-D, NAA, IBA, and IAA.This result suggests that the reduced sensitivity to exogenous auxins may be due tothe altered auxin activity in the root, thereby affecting root morphology inLrt1.     

Shoot-derived auxin is essential for early lateral root emergence in Arabidopsis seedlings

Lateral root formation is profoundly affected by auxins. Here we present data which indicate that light influences the formation of indole-3-acetic acid (IAA) in germinating Arabidopsis seedlings. IAA transported from the developing leaves to the root system is detectable as a short-lived pulse in the roots and is required for the emergence of the lateral root primordia (LRP) during early seedling development. LRP emergence is inhibited by the removal of apical tissues prior to detection of the IAA pulse in the root, but this treatment has minimal effects on LRP initiation. Our results identify the first developing true leaves as the most likely source for the IAA required for the first emergence of the LRP, as removal of cotyledons has only a minor effect on LRP emergence in contrast to removal of the leaves. A basipetal IAA concentration gradient with high levels of IAA in the root tip appears to control LRP initiation, in contrast to their emergence. A significant increase in the ability of the root system to synthesize IAA is observed 10 days after germination, and this in turn is reflected in the reduced dependence of the lateral root emergence on aerial tissue-derived auxin at this stage. We propose a model for lateral root formation during early seedling development that can be divided into two phases: (i) an LRP initiation phase dependent on a root tip-localized IAA source, and (ii) an LRP emergence phase dependent on leaf-derived IAA up to 10 days after germination.     

Studies on sodium bypass flow in lateral rootless mutants lrt1 and lrt2, and crown rootless mutant crl1 of rice (Oryza sativa L.)

An apoplastic pathway, the so‐called bypass flow, is important for Na⁺ uptake in rice (Oryza sativa L.) under saline conditions; however, the precise site of entry is not yet known. We report the results of our test of the hypothesis that bypass flow of Na⁺ in rice occurs at the site where lateral roots emerge from the main roots. We investigated Na⁺ uptake and bypass flow in lateral rootless mutants (lrt1, lrt2), a crown rootless mutant (crl1), their wild types (Oochikara, Nipponbare and Taichung 65, respectively) and in seedlings of rice cv. IR36. The results showed that shoot Na⁺ concentration in lrt1, lrt2 and crl1 was lower (by 20–23%) than that of their wild types. In contrast, the bypass flow quantified using trisodium‐8‐hydroxy‐1,3,6‐pyrenetrisulphonic acid (PTS) was significantly increased in the mutants, from an average of 1.1% in the wild types to 3.2% in the mutants. Similarly, bypass flow in shoots of IR36 where the number of lateral and crown roots had been reduced through physical and hormonal manipulations was dramatically increased (from 5.6 to 12.5%) as compared to the controls. The results suggest that the path of bypass flow in rice is not at the sites of lateral root emergence.     

CRD1, an Xpo1 domain protein,regulates miRNA accumulation and crown root development in rice

Crown root (CR) is the main component of the fibrous root system in cereal crops, but the molecular mechanism underlying CR development is still unclear. Here, we isolated the crown root defect 1 (crd1) mutant from ethyl methane sulfonate‐mutated mutant library, which significantly inhibited CR development. The CRD1 was identified through genome resequencing and complementation analysis, which encodes an Xpo1 domain protein: the rice ortholog of Arabidopsis HASTY (HST) and human exportin‐5 (XPO5). CRD1 is ubiquitously expressed, with the highest expression levels in the CR primordium at the stem base. CRD1 is a nucleocytoplasmic protein. The crd1 mutant contains significantly reduced miRNA levels in the cytoplasm and nucleus, suggesting that CRD1 is essential for maintaining normal miRNA levels in plant cells. The altered CR phenotype of crd1 was simulated by target mimicry of miR156, suggesting that this defect is due to the disruption of miR156 regulatory pathways. Our analysis of CRD1, the HST ortholog identified in monocots, expands our understanding of the molecular mechanisms underlying miRNA level and CR development.     

ARL1, a LOB-domain protein required for adventitious root formation in rice

Adventitious roots constitute the bulk of the fibrous root system in cereals. Compared with the current understanding of shoot development, knowledge of the molecular mechanisms of development of the adventitious roots of cereals is limited. We have isolated and characterized a novel gene controlling the initiation of adventitious root primordia in rice (Oryza sativa L.). The gene, designated Adventitious rootless1 (ARL1), encodes a protein with a LATERAL ORGAN BOUNDARIES (LOB) domain. It is expressed in lateral and adventitious root primordia, tiller primordia, vascular tissues, scutellum, and young pedicels. ARL1 is a nuclear protein and can form homodimers. ARL1 is an auxin- and ethylene-responsive gene, and the expression pattern of ARL1 in roots parallels auxin distribution. Our findings suggest that ARL1 is an auxin-responsive factor involved in auxin-mediated cell dedifferentiation, and that it promotes the initial cell division in the pericycle cells adjacent to the peripheral vascular cylinder in the stem.     

Ubiquitin-dependent Argonauteprotein MEL1 degradation is essential for rice sporogenesis and phasiRNA target regulation

MEIOSIS ARRESTED AT LEPTOTENE1 (MEL1), a rice (Oryza sativa) Argonaute (AGO) protein, has been reported to function specifically at premeiotic and meiotic stages of germ cell development and is associated with a novel class of germ cell-specific small noncoding RNAs called phased small RNAs (phasiRNAs). MEL1 accumulation is temporally and spatially regulated and is eliminated after meiosis. However, the metabolism and turnover (i.e. the homeostasis) of MEL1 during germ cell development remains unknown. Here, we show that MEL1 is ubiquitinated and subsequently degraded via the proteasome pathway in vivo during late sporogenesis. Abnormal accumulation of MEL1 after meiosis leads to a semi-sterile phenotype. We identified a monocot-specific E3 ligase, XBOS36, a CULLIN RING-box protein, that is responsible for the degradation of MEL1. Ubiquitination at four K residues at the N terminus of MEL1 by XBOS36 induces its degradation. Importantly, inhibition of MEL1 degradation either by XBOS36 knockdown or by MEL1 overexpression prevents the formation of pollen at the microspore stage. Further mechanistic analysis showed that disrupting MEL1 homeostasis in germ cells leads to off-target cleavage of phasiRNA target genes. Our findings thus provide insight into the communication between a monocot-specific E3 ligase and an AGO protein during plant reproductive development.

The degradation of Argonaute protein MEL1 is mediated by a monocot-specific E3 ubiquitin ligase XBOS36 to avoid off-target regulation of phasiRNAs during rice sporogenesis.     

OsAGAP, an ARF-GAP from rice, regulates root development mediated by auxin in Arabidopsis

Arf (ADP-ribosylation factor) proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity. They rely on the action of GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs) for their function. In the present study the OsAGAP gene in rice, which encoded a protein with predicted structure similar to ArfGAP, was identified. The purified OsAGAP-GST fusion protein was able to stimulate the GTPase activity of rice Arf. Furthermore, OsAGAP can rescue the defect of vesicular transport in the yeast gcs1 delta glo3 delta double-mutant cells. Transgenic Arabidopsis with OsAGAP constitutively expression showed reduced apical dominance, shorter primary roots, increasing number of longer adventitious roots. Many of the phenotypes can be phenocopied by treatment of exogenous indoleacetic acid level (IAA) in wild-type plants. Determination of whole-plant IAA level showed that there is a sharp increase of free IAA in OsAGAP transgenic Arabidopsis seedlings. In addition, removal of the 4-day-old shoot apex could inhibit the adventitious root formation in the transgenic seedlings. These results suggest OsAGAP, an ARF-GAP of rice, maybe involved in the mediation of plant root development by regulating auxin level.