Functional characterization of hydroxyproline-O-galactosyltransferases for Arabidopsis arabinogalactan-protein synthesis.

BACKGROUND: Arabinogalactan-proteins (AGPs) are structurally complex hydroxyproline-rich cell wall glycoproteins ubiquitous in the plant kingdom. AGPs biosynthesis involves a series of post-translational modifications including the addition of type II arabinogalactans to non-contiguous Hyp residues. To date, eight Hyp-galactosyltransferases (Hyp-GALTs; GALT2-GALT9) belonging to CAZy GT31, are known to catalyze the addition of the first galactose residues to AGP protein backbones and enable subsequent AGP glycosylation. The extent of genetic redundancy, however, remains to be elucidated for the Hyp-GALT gene family. RESULTS: To examine their gene redundancy and functions, we generated various multiple gene knock-outs, including a triple mutant (galt5 galt8 galt9), two quadruple mutants (galt2 galt5 galt7 galt8, galt2 galt5 galt7 galt9), and one quintuple mutant (galt2 galt5 galt7 galt8 galt9), and comprehensively examined their biochemical and physiological phenotypes. The key findings include: AGP precipitations with ß-Yariv reagent showed that GALT2, GALT5, GALT7, GALT8 and GALT9 act redundantly with respect to AGP glycosylation in cauline and rosette leaves, while the activity of GALT7, GALT8 and GALT9 dominate in the stem, silique and flowers. Monosaccharide composition analysis showed that galactose was decreased in the silique and root AGPs of the Hyp-GALT mutants. TEM analysis of 25789 quintuple mutant stems indicated cell wall defects coincident with the observed developmental and growth impairment in these Hyp-GALT mutants. Correlated with expression patterns, galt2, galt5, galt7, galt8, and galt9 display equal additive effects on insensitivity to ß-Yariv-induced growth inhibition, silique length, plant height, and pollen viability. Interestingly, galt7, galt8, and galt9 contributed more to primary root growth and root tip swelling under salt stress, whereas galt2 and galt5 played more important roles in seed morphology, germination defects and seed set. Pollen defects likely contributed to the reduced seed set in these mutants. CONCLUSION: Additive and pleiotropic effects of GALT2, GALT5, GALT7, GALT8 and GALT9 on vegetative and reproductive growth phenotypes were teased apart via generation of different combinations of Hyp-GALT knock-out mutants. Taken together, the generation of higher order Hyp-GALT mutants demonstrate the functional importance of AG polysaccharides decorating the AGPs with respect to various aspects of plant growth and development.

The DME demethylase regulates sporophyte gene expression, cell proliferation, differentiation, and meristem resurrection.

The flowering plant life cycle consists of alternating haploid (gametophyte) and diploid (sporophyte) generations, where the sporophytic generation begins with fertilization of haploid gametes. In Arabidopsis, genome-wide DNA demethylation is required for normal development, catalyzed by the DEMETER (DME) DNA demethylase in the gamete companion cells of male and female gametophytes. In the sporophyte, postembryonic growth and development are largely dependent on the activity of numerous stem cell niches, or meristems. Analyzing Arabidopsis plants homozygous for a loss-of-function dme-2 allele, we show that DME influences many aspects of sporophytic growth and development. dme-2 mutants exhibited delayed seed germination, variable root hair growth, aberrant cellular proliferation and differentiation followed by enhanced de novo shoot formation, dysregulation of root quiescence and stomatal precursor cells, and inflorescence meristem (IM) resurrection. We also show that sporophytic DME activity exerts a profound effect on the transcriptome of developing Arabidopsis plants, including discrete groups of regulatory genes that are misregulated in dme-2 mutant tissues, allowing us to potentially link phenotypes to changes in specific gene expression pathways. These results show that DME plays a key role in sporophytic development and suggest that DME-mediated active DNA demethylation may be involved in the maintenance of stem cell activities during the sporophytic life cycle in Arabidopsis.

An Acyl-CoA N-Acyltransferase Regulates Meristem Phase Change and Plant Architecture in Barley.

The modification of shoot architecture and increased investment into reproductive structures is key for crop improvement and is achieved through coordinated changes in the development and determinacy of different shoot meristems. A fundamental question is how the development of different shoot meristems is genetically coordinated to optimize the balance between vegetative and reproductive organs. Here we identify the MANY NODED DWARF1 (HvMND1) gene as a major regulator of plant architecture in barley (Hordeum vulgare). The mnd1.a mutant displayed an extended vegetative program with increased phytomer, leaf, and tiller production but a reduction in the number and size of grains. The induction of vegetative structures continued even after the transition to reproductive growth, resulting in a marked increase in longevity. Using mapping by RNA sequencing, we found that the HvMND1 gene encodes an acyl-CoA N-acyltransferase that is predominately expressed in developing axillary meristems and young inflorescences. Exploration of the expression network modulated by HvMND1 revealed differential expression of the developmental microRNAs miR156 and miR172 and several key cell cycle and developmental genes. Our data suggest that HvMND1 plays a significant role in the coordinated regulation of reproductive phase transitions, thereby promoting reproductive growth and whole plant senescence in barley.

Cell Wall Invertase Is Essential for Ovule Development through Sugar Signaling Rather Than Provision of Carbon Nutrients.

Ovule formation is essential for realizing crop yield because it determines seed number. The underlying molecular mechanism, however, remains elusive. Here, we show that cell wall invertase (CWIN) functions as a positive regulator of ovule initiation in Arabidopsis (Arabidopsis thaliana). In situ hybridization revealed that CWIN2 and CWIN4 were expressed at the placenta region where ovule primordia initiated. Specific silencing of CWIN2 and CWIN4 using targeted artificial microRNA driven by an ovule-specific SEEDSTICK promoter (pSTK) resulted in a substantial reduction of CWIN transcript and activity, which blocked ovule initiation and aggravated ovule abortion. There was no induction of carbon (C) starvation genes in the transgenic lines, and supplementing newly forming floral buds with extra C failed to recover the ovule phenotype. This indicates that suppression of CWIN did not lead to C starvation. A group of hexose transporters was downregulated in the transgenic plants. Among them, two representative ones were spatially coexpressed with CWIN2 and CWIN4, suggesting a coupling between CWIN and hexose transporters for ovule initiation. RNA-sequencing analysis identified differentially expressed genes encoding putative extracellular receptor-like kinases, MADS-box transcription factors, including STK, and early auxin response genes in response to CWIN-silencing. Our data demonstrate the essential role of CWIN in ovule initiation, which is most likely to occur through sugar signaling instead of C nutrient contribution. We propose that CWIN-mediated sugar signaling may be perceived by, and transmitted through, hexose transporters or receptor-like kinases to regulate ovule formation by modulating downstream auxin signaling and MADS-box transcription factors.

Xyloglucans and Microtubules Synergistically Maintain Meristem Geometry and Phyllotaxis.

The shoot apical meristem (SAM) gives rise to all aerial plant organs. Cell walls are thought to play a central role in this process, translating molecular regulation into dynamic changes in growth rate and direction, although their precise role in morphogenesis during organ formation is poorly understood. Here, we investigated the role of xyloglucans (XyGs), a major, yet functionally poorly characterized, wall component in the SAM of Arabidopsis (Arabidopsis thaliana). Using immunolabeling, biochemical analysis, genetic approaches, microindentation, laser ablation, and live imaging, we showed that XyGs are important for meristem shape and phyllotaxis. No difference in the Young's modulus (i.e. an indicator of wall stiffness) of the cell walls was observed when XyGs were perturbed. Mutations in enzymes required for XyG synthesis also affect other cell wall components such as cellulose content and pectin methylation status. Interestingly, control of cortical microtubule dynamics by the severing enzyme KATANIN became vital when XyGs were perturbed or absent. This suggests that the cytoskeleton plays an active role in compensating for altered cell wall composition.

An Aminoacyl tRNA Synthetase, OKI1, Is Required for Proper Shoot Meristem Size in Arabidopsis.

In plants, the stem cells that form the shoot system reside within the shoot apical meristem (SAM), which is regulated by feedback signaling between the WUSCHEL (WUS) homeobox protein and CLAVATA (CLV) peptides and receptors. WUS-CLV feedback signaling can be modulated by various endogenous or exogenous factors, such as chromatin state, hormone signaling, reactive oxygen species (ROS) signaling and nutrition, leading to a dynamic control of SAM size corresponding to meristem activity. Despite these insights, however, the knowledge of genes that control SAM size is still limited, and in particular, the regulation by ROS signaling is only beginning to be comprehended. In this study, we report a new function in maintenance of SAM size, encoded by the OKINA KUKI1 (OKI1) gene. OKI1 is expressed in the SAM and encodes a mitochondrial aspartyl tRNA synthetase (AspRS). oki1 mutants display enlarged SAMs with abnormal expression of WUS and CLV3 and overaccumulation of ROS in the meristem. Our findings support the importance of normal AspRS function in the maintenance of the WUS-CLV3 feedback loop and SAM size.

Mitogen-activated protein kinase 6 integrates phosphate and iron responses for indeterminate root growth in Arabidopsis thaliana.

MAIN CONCLUSION: A MAPK module, of which MPK6 kinase is an important component, is involved in the coordination of the responses to Pi and Fe in the primary root meristem of Arabidopsis thaliana. Phosphate (Pi) deficiency induces determinate primary root growth in Arabidopsis through cessation of cell division in the meristem, which is linked to an increased iron (Fe) accumulation. Here, we show that Mitogen-Activated Protein Kinase6 (MPK6) has a role in Arabidopsis primary root growth under low Pi stress. MPK6 activity is induced in roots in response to low Pi, and such induction is enhanced by Fe supplementation, suggesting an MPK6 role in coordinating Pi/Fe balance in mediating root growth. The differentiation of the root meristem induced by low Pi levels correlates with altered expression of auxin-inducible genes and auxin transporter levels via MPK6. Our results indicate a critical role of the MPK6 kinase in coordinating meristem cell activity to Pi and Fe availability for proper primary root growth.

Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth.

How organisms attain their specific shapes and modify their growth patterns in response to environmental and chemical signals has been the subject of many investigations. Plant cells are at high turgor pressure and are surrounded by a rigid yet flexible cell wall, which is the primary determinant of plant growth and morphogenesis. Cellulose microfibrils, synthesized by plasma membrane-localized cellulose synthase complexes, are major tension-bearing components of the cell wall that mediate directional growth. Despite advances in understanding the genetic and biophysical regulation of morphogenesis, direct studies of cellulose biosynthesis and its impact on morphogenesis of different cell and tissue types are largely lacking. In this study, we took advantage of mutants of three primary cellulose synthase (CESA) genes that are involved in primary wall cellulose synthesis. Using field emission scanning electron microscopy, live cell imaging and biophysical measurements, we aimed to understand how the primary wall CESA complex acts during shoot apical meristem development. Our results indicate that cellulose biosynthesis impacts the mechanics and growth of the shoot apical meristem.

A wheat/rye polymorphism affects seminal root length and yield across different irrigation regimes.

The introgression of a small segment of wheat (Triticum aestivum L.) chromosome arm 1BS in the distal region of the rye (Secale cereale L.) 1RS.1BL arm translocation in wheat (henceforth 1RSRW) was previously associated with reduced grain yield, carbon isotope discrimination, and stomatal conductance, suggesting reduced access to soil moisture. Here we show that lines with the normal 1RS arm have longer roots than lines with the 1RSRW arm in both field and hydroponic experiments. In the 1RSRW lines, differences in seminal root length were associated with a developmentally regulated arrest of the root apical meristem (RAM). Approximately 10 d after germination, the seminal roots of the 1RSRW plants showed a gradual reduction in elongation rate, and stopped growing a week later. Seventeen days after germination, the roots of the 1RSRW plants showed altered gradients of reactive oxygen species and emergence of lateral roots close to the RAM, suggesting changes in the root meristem. The 1RSRW lines also showed reduced biomass (estimated by the normalized difference vegetation index) and grain yield relative to the 1RS lines, with larger differences under reduced or excessive irrigation than under normal irrigation. These results suggest that this genetic variation could be useful to modulate root architecture.

Control of meristem determinacy by trehalose 6-phosphate phosphatases is uncoupled from enzymatic activity.

Meristem fate is regulated by trehalose 6-phosphate phosphatases (TPPs), but their mechanism of action remains mysterious. Loss of the maize TPPs RAMOSA3 and TPP4 leads to reduced meristem determinacy and more inflorescence branching. However, analysis of an allelic series revealed no correlation between enzymatic activity and branching, and a catalytically inactive version of RA3 complements the ra3 mutant. Together with their nuclear localization, these findings suggest a moonlighting function for TPPs.

A TOR-YAK1 signaling axis controls cell cycle, meristem activity and plant growth in Arabidopsis.

TARGET OF RAPAMYCIN (TOR) is a conserved eukaryotic phosphatidylinositol-3-kinase-related kinase that plays a major role in regulating growth and metabolism in response to environment in plants. We performed a genetic screen for Arabidopsis ethylmethane sulfonate mutants resistant to the ATP-competitive TOR inhibitor AZD-8055 to identify new components of the plant TOR pathway. We found that loss-of-function mutants of the DYRK (dual specificity tyrosine phosphorylation regulated kinase)/YAK1 kinase are resistant to AZD-8055 and, reciprocally, that YAK1 overexpressors are hypersensitive to AZD-8055. Significantly, these phenotypes were conditional on TOR inhibition, positioning YAK1 activity downstream of TOR. We further show that the ATP-competitive DYRK1A inhibitor pINDY phenocopies YAK1 loss of function. Microscopy analysis revealed that YAK1 functions to repress meristem size and induce differentiation. We show that YAK1 represses cyclin expression in the different zones of the root meristem and that YAK1 is essential for TOR-dependent transcriptional regulation of the plant-specific SIAMESE-RELATED (SMR) cyclin-dependent kinase inhibitors in both meristematic and differentiating root cells. Thus, YAK1 is a major regulator of meristem activity and cell differentiation downstream of TOR.

Epigenetic reprogramming by histone acetyltransferase HAG1/AtGCN5 is required for pluripotency acquisition in Arabidopsis.

Shoot regeneration can be achieved in vitro through a two-step process involving the acquisition of pluripotency on callus-induction media (CIM) and the formation of shoots on shoot-induction media. Although the induction of root-meristem genes in callus has been noted recently, the mechanisms underlying their induction and their roles in de novo shoot regeneration remain unanswered. Here, we show that the histone acetyltransferase HAG1/AtGCN5 is essential for de novo shoot regeneration. In developing callus, it catalyzes histone acetylation at several root-meristem gene loci including WOX5, WOX14, SCR, PLT1, and PLT2, providing an epigenetic platform for their transcriptional activation. In turn, we demonstrate that the transcription factors encoded by these loci act as key potency factors conferring regeneration potential to callus and establishing competence for de novo shoot regeneration. Thus, our study uncovers key epigenetic and potency factors regulating plant-cell pluripotency. These factors might be useful in reprogramming lineage-specified plant cells to pluripotency.

OsPIN1b is Involved in Rice Seminal Root Elongation by Regulating Root Apical Meristem Activity in Response to Low Nitrogen and Phosphate.

The response of plant root development to nutrient deficiencies is critical for crop production. Auxin, nitric oxide (NO), and strigolactones (SLs) are important regulators of root growth under low-nitrogen and -phosphate (LN and LP) conditions. Polar auxin transport in plants, which is mainly dependent on auxin efflux protein PINs, creates local auxin maxima to form the basis for root initiation and elongation; however, the PIN genes that play an important role in LN- and LP-modulated root growth remain unclear. qRT-PCR analysis of OsPIN family genes showed that the expression of OsPIN1b is most abundant in root tip and is significantly downregulated by LN, LP, sodium nitroprusside (SNP, NO donor), and GR24 (analogue of SLs) treatments. Seminal roots in ospin1b mutants were shorter than those of the wild type; and the seminal root, [3H]IAA transport, and IAA concentration responses to LN, LP, SNP, and GR24 application were attenuated in ospin1b-1 mutants. pCYCB1;1::GUS expression was upregulated by LN, LP, SNP, and GR24 treatments in wild type, but not in the ospin1b-1 mutant, suggesting that OsPIN1b is involved in auxin transport and acts as a downstream mediator of NO and SLs to induce meristem activity in root tip in rice under LN and LP.

Mitogen activated protein kinase 6 and MAP kinase phosphatase 1 are involved in the response of Arabidopsis roots to L-glutamate.

KEY MESSAGE: The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.

Knockdown of an inflorescence meristem-specific cytokinin oxidase - OsCKX2 in rice reduces yield penalty under salinity stress condition.

Cytokinins play a significant role in determining grain yield in plants. Cytokinin oxidases catalyse irreversible degradation of cytokinins and hence modulate cellular cytokinin levels. Here, we studied the role of an inflorescence meristem-specific rice cytokinin oxidase - OsCKX2 - in reducing yield penalty under salinity stress conditions. We utilized an RNAi-based approach to study the function of OsCKX2 in maintaining grain yield under salinity stress condition. Ultra-performance liquid chromatography-based estimation revealed a significant increase in cytokinins in the inflorescence meristem of OsCKX2-knockdown plants. To determine if there exists a correlation between OsCKX2 levels and yield under salinity stress condition, we assessed the growth, physiology and grain yield of OsCKX2-knockdown plants vis-à-vis the wild type. OsCKX2-knockdown plants showed better vegetative growth, higher relative water content and photosynthetic efficiency and reduced electrolyte leakage as compared with the wild type under salinity stress. Importantly, we found a negative correlation between OsCKX2 expression and plant productivity as evident by assessment of agronomical parameters such as panicle branching, filled grains per plant and harvest index both under control and salinity stress conditions. These results suggest that OsCKX2, via controlling cytokinin levels, regulates floral primordial activity modulating rice grain yield under normal as well as abiotic stress conditions.

Plant-Specific Histone Deacetylases HDT1/2 Regulate GIBBERELLIN 2-OXIDASE2 Expression to Control Arabidopsis Root Meristem Cell Number.

Root growth is modulated by environmental factors and depends on cell production in the root meristem (RM). New cells in the meristem are generated by stem cells and transit-amplifying cells, which together determine RM cell number. Transcription factors and chromatin-remodeling factors have been implicated in regulating the switch from stem cells to transit-amplifying cells. Here, we show that two Arabidopsis thaliana paralogs encoding plant-specific histone deacetylases, HDT1 and HDT2, regulate a second switch from transit-amplifying cells to expanding cells. Knockdown of HDT1/2 (hdt1,2i) results in an earlier switch and causes a reduced RM cell number. Our data show that HDT1/2 negatively regulate the acetylation level of the C19-GIBBERELLIN 2-OXIDASE2 (GA2ox2) locus and repress the expression of GA2ox2 in the RM and elongation zone. Overexpression of GA2ox2 in the RM phenocopies the hdt1,2i phenotype. Conversely, knockout of GA2ox2 partially rescues the root growth defect of hdt1,2i These results suggest that by repressing the expression of GA2ox2, HDT1/2 likely fine-tune gibberellin metabolism and they are crucial for regulating the switch from cell division to expansion to determine RM cell number. We propose that HDT1/2 function as part of a mechanism that modulates root growth in response to environmental factors.

Cytosolic glyceraldehyde-3-phosphate dehydrogenases play crucial roles in controlling cold-induced sweetening and apical dominance of potato (Solanum tuberosum L.) tubers.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an important enzyme that functions in producing energy and supplying intermediates for cellular metabolism. Recent researches indicate that GAPDHs have multiple functions beside glycolysis. However, little information is available for functions of GAPDHs in potato. Here, we identified 4 putative cytosolic GAPDH genes in potato genome and demonstrated that the StGAPC1, StGAPC2, and StGAPC3, which are constitutively expressed in potato tissues and cold inducible in tubers, encode active cytosolic GAPDHs. Cosuppression of these 3 GAPC genes resulted in low tuber GAPDH activity, consequently the accumulation of reducing sugars in cold stored tubers by altering the tuber metabolite pool sizes favoring the sucrose pathway. Furthermore, GAPCs-silenced tubers exhibited a loss of apical dominance dependent on cell death of tuber apical bud meristem (TAB-meristem). It was also confirmed that StGAPC1, StGAPC2, and StGAPC3 interacted with the autophagy-related protein 3 (ATG3), implying that the occurrence of cell death in TAB-meristem could be induced by ATG3 associated events. Collectively, the present research evidences first that the GAPC genes play crucial roles in diverse physiological and developmental processes in potato tubers.

Overexpression of DEMETER, a DNA demethylase, promotes early apical bud maturation in poplar.

The transition from active growth to dormancy is critical for the survival of perennial plants. We identified a DEMETER-like (CsDML) cDNA from a winter-enriched cDNA subtractive library in chestnut (Castanea sativa Mill.), an economically and ecologically important species. Next, we characterized this DNA demethylase and its putative ortholog in the more experimentally tractable hybrid poplar (Populus tremula × alba), under the signals that trigger bud dormancy in trees. We performed phylogenetic and protein sequence analysis, gene expression profiling, and 5-methyl-cytosine methylation immunodetection studies to evaluate the role of CsDML and its homolog in poplar, PtaDML6. Transgenic hybrid poplars overexpressing CsDML were produced and analysed. Short days and cold temperatures induced CsDML and PtaDML6. Overexpression of CsDML accelerated short-day-induced bud formation, specifically from Stages 1 to 0. Buds acquired a red-brown coloration earlier than wild-type plants, alongside with the up-regulation of flavonoid biosynthesis enzymes and accumulation of flavonoids in the shoot apical meristem and bud scales. Our data show that the CsDML gene induces bud formation needed for the survival of the apical meristem under the harsh conditions of winter.

Vacuolar processing enzyme activates programmed cell death in the apical meristem inducing loss of apical dominance.

The potato (Solanum tuberosum L.) tuber is a swollen underground stem that can sprout in an apical dominance (AD) pattern. Bromoethane (BE) induces loss of AD and the accumulation of vegetative vacuolar processing enzyme (S. tuberosum vacuolar processing enzyme [StVPE]) in the tuber apical meristem (TAM). Vacuolar processing enzyme activity, induced by BE, is followed by programmed cell death in the TAM. In this study, we found that the mature StVPE1 (mVPE) protein exhibits specific activity for caspase 1, but not caspase 3 substrates. Optimal activity of mVPE was achieved at acidic pH, consistent with localization of StVPE1 to the vacuole, at the edge of the TAM. Downregulation of StVPE1 by RNA interference resulted in reduced stem branching and retained AD in tubers treated with BE. Overexpression of StVPE1 fused to green fluorescent protein showed enhanced stem branching after BE treatment. Our data suggest that, following stress, induction of StVPE1 in the TAM induces AD loss and stem branching.

ZYGOTE-ARREST 3 that encodes the tRNA ligase is essential for zygote division in Arabidopsis.

In sexual organisms, division of the zygote initiates a new life cycle. Although several genes involved in zygote division are known in plants, how the zygote is activated to start embryogenesis has remained elusive. Here, we showed that a mutation in ZYGOTE-ARREST 3 (ZYG3) in Arabidopsis led to a tight zygote-lethal phenotype. Map-based cloning revealed that ZYG3 encodes the transfer RNA (tRNA) ligase AtRNL, which is a single-copy gene in the Arabidopsis genome. Expression analyses showed that AtRNL is expressed throughout zygotic embryogenesis, and in meristematic tissues. Using pAtRNL::cAtRNL-sYFP-complemented zyg3/zyg3 plants, we showed that AtRNL is localized exclusively in the cytoplasm, suggesting that tRNA splicing occurs primarily in the cytoplasm. Analyses using partially rescued embryos showed that mutation in AtRNL compromised splicing of intron-containing tRNA. Mutations of two tRNA endonuclease genes, SEN1 and SEN2, also led to a zygote-lethal phenotype. These results together suggest that tRNA splicing is critical for initiating zygote division in Arabidopsis.