When attacked by a phloem-feeding greenbug aphid (Schizaphis graminum), sorghum (Sorghum bicolor) activates jasmonic acid (JA)- and salicylic acid (SA)-regulated genes, as well as genes outside known wounding and SA signaling pathways. A collection of 672 cDNAs was obtained by differential subtraction with cDNAs prepared from sorghum seedlings infested by greenbug aphids and those from uninfested seedlings. Subsequent expression profiling using DNA microarray and northern-blot analyses identified 82 transcript types from this collection responsive to greenbug feeding, methyl jasmonate (MeJA), or SA application. DNA sequencing analyses indicated that these encoded proteins functioning in direct defense, defense signaling, oxidative burst, secondary metabolism, abiotic stress, cell maintenance, and photosynthesis, as well as proteins of unknown function. In response to insect feeding, sorghum increased transcript abundance of numerous defense genes, with some SA-dependent pathogenesis-related genes responding to greenbug more strongly than to SA. In contrast, only weak induction of MeJA-regulated defense genes was observed after greenbug treatment. However, infestation tests confirmed that JA-regulated pathways were effective in plant defense against greenbugs. Activation of certain transcripts exclusively by greenbug infestation was observed, and may represent unique signal transduction events independent of JA- and SA-regulated pathways. Results indicate that plants coordinately regulate defense gene expression when attacked by phloem-feeding aphids, but also suggest that aphids are able to avoid triggering activation of some otherwise potentially effective plant defensive machinery, possibly through their particular mode of feeding.

Two Arabidopsis thaliana extragenic mutations that suppress NaCl hypersensitivity of the sos3-1 mutant were identified in a screen of a T-DNA insertion population in the genetic background of Col-0 gl1 sos3-1. Analysis of the genome sequence in the region flanking the T-DNA left border indicated that sos3-1 hkt1-1 and sos3-1 hkt1-2 plants have allelic mutations in AtHKT1. AtHKT1 mRNA is more abundant in roots than shoots of wild-type plants but is not detected in plants of either mutant, indicating that this gene is inactivated by the mutations. hkt1-1 and hkt1-2 mutations can suppress to an equivalent extent the Na ؉ sensitivity of sos3-1 seedlings and reduce the intracellular accumulation of this cytotoxic ion. Moreover, sos3-1 hkt1-1 and sos3-1 hkt1-2 seedlings are able to maintain [K ؉ ]int in medium supplemented with NaCl and exhibit a substantially higher intracellular ratio of K ؉ ͞Na ؉ than the sos3-1 mutant. Furthermore, the hkt1 mutations abrogate the growth inhibition of the sos3-1 mutant that is caused by K ؉ deficiency on culture medium with low Ca 2؉ (0.15 mM) and <200 M K ؉ . Interestingly, the capacity of hkt1 mutations to suppress the Na ؉ hypersensitivity of the sos3-1 mutant is reduced substantially when seedlings are grown in medium with low Ca 2؉ (0.15 mM). These results indicate that AtHKT1 is a salt tolerance determinant that controls Na ؉ entry and high affinity K ؉ uptake. The hkt1 mutations have revealed the existence of another Na ؉ influx system(s) whose activity is reduced by high [Ca 2؉ ]ext. H igh [NaCl] ext disturbs intracellular ion homeostasis of plants, which leads to membrane dysfunction, attenuation of metabolic activity, and secondary effects that cause growth inhibition and lead ultimately to cell death (1). Both glycophytes and halophytes use a similar strategy that involves regulation of net Na ϩ flux across the plasma membrane and vacuolar compartmentalization of the internalized cation to mediate intracellular Na ϩ homeostasis. This strategy requires the coordinated function of numerous ion transport determinants and effectively partitions the toxic ion away from critical cytosolic and organellar machinery. Under conditions of high [Na ϩ ] ext , the functioning of these determinants also facilitates the use of Na ϩ as an osmolyte to mediate osmotic adjustment that is necessary for cell expansion (1-3). Because vacuolar expansion is the primary mechanism of plant cell enlargement, this strategy is likely to be an essential adaptation to saline environments.Recently, putative plasma membrane and tonoplast localized Na ϩ ͞H ϩ transporters were identified in plants that are presumed to mediate energized transport of Na ϩ outward from the cytosol to the apoplast or into the vacuole (4-7). These transporters are apparently the molecular effectors of Na ϩ ͞H ϩ antiporter activities associated with plasma membrane and tonoplast vesicles that were described more than a decade ago (1,3,8,9). The plasma membrane Na ϩ ͞H ϩ

Arabidopsis stt3a-1 and stt3a-2 mutations cause NaCl/osmotic sensitivity that is characterized by reduced cell division in the root meristem. Sequence comparison of the STT3a gene identified a yeast ortholog, STT3 , which encodes an essential subunit of the oligosaccharyltransferase complex that is involved in protein N -glycosylation. NaCl induces the unfolded protein response in the endoplasmic reticulum (ER) and cell cycle arrest in root tip cells of stt3a seedlings, as determined by expression profiling of ER stress-responsive chaperone ( BiP -GUS ) and cell division ( CycB1;1 -GUS ) genes, respectively. Together, these results indicate that plant salt stress adaptation involves ER stress signal regulation of cell cycle progression. Interestingly, a mutation ( stt3b-1 ) in another Arabidopsis STT3 isogene ( STT3b ) does not cause NaCl sensitivity. However, the stt3a-1 stt3b-1 double mutation is gametophytic lethal. Apparently, STT3a and STT3b have overlapping and essential functions in plant growth and developmental processes, but the pivotal and specific protein glycosylation that is a necessary for recovery from the unfolded protein response and for cell cycle progression during salt/osmotic stress recovery is associated uniquely with the function of the STT3a isoform.

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Proteins that specifically bind double-stranded RNA (dsRNA) are involved in the regulation of cellular signaling events and gene expression, and are characterized by a conserved dsRNA-binding motif (dsRBM). Here we report the biochemical properties of nine such gene products, each containing one or two dsRBMs: four Arabidopsis Dicer-like proteins (DCL1-4), Arabidopsis HYL1 and four of its homologs (DRB2, DRB4, DRB5 and OsDRB1). DCL1, DCL3, HYL1 and the four HYL1 homologs exhibit significant dsRNA-binding activity, indicating that these proteins are involved in RNA metabolism. The dsRBMs from dsRBM-containing proteins (dsRBPs) also function as a protein-protein interaction domain and homo- and heterodimerization are essential for biological functioning of these proteins. We show that DRB4 interacts specifically with DCL4, and HYL1 most strongly interacts with DCL1. These results indicate that each HYL1/DRB family protein interacts with one specific partner among the four Dicer-like proteins. Localization studies using GFP fusion proteins demonstrate that DCL1, DCL4, HYL1 and DRB4 localize in the nucleus, while DRB2 is present in the cytoplasm. Subcellular localizations of HYL1, DRB4, DCL1 and DCL4 further strengthen the notion that HYL1 and DCL1, and DRB4 and DCL4, exist as complexes. The presented data suggest that each member of the HYL1/DRB protein family may individually modulate Dicer function through heterodimerization with a Dicer-like protein in vivo.

Protein N -glycosylation in the endoplasmic reticulum (ER) and in the Golgi apparatus is an essential process in eukaryotic cells. Although the N -glycosylation pathway in the ER has been shown to regulate protein quality control, salt tolerance, and cellulose biosynthesis in plants, no biological roles have been linked functionally to N -glycan modifications that occur in the Golgi apparatus. Herein, we provide evidence that mutants defective in N -glycan maturation, such as complex glycan 1 ( cgl1 ), are more salt-sensitive than wild type. Salt stress caused growth inhibition, aberrant root-tip morphology, and callose accumulation in cgl1 , which were also observed in an ER oligosaccharyltransferase mutant, staurosporin and temperature sensitive 3a ( stt3a ). Unlike stt3a , cgl1 did not cause constitutive activation of the unfolded protein response. Instead, aberrant modification of the plasma membrane glycoprotein KORRIGAN 1/RADIALLY SWOLLEN 2 (KOR1/RSW2) that is necessary for cellulose biosynthesis occurred in cgl1 and stt3a . Genetic analyses identified specific interactions among rsw2 , stt3a , and cgl1 mutations, indicating that the function of KOR1/RSW2 protein depends on complex N -glycans. Furthermore, cellulose deficient rsw1-1 and rsw2-1 plants were also salt-sensitive. These results establish that plant protein N -glycosylation functions beyond protein folding in the ER and is necessary for sufficient cell-wall formation under salt stress.

N-Glycans attached to the ectodomains of plasma membrane pattern recognition receptors constitute likely initial contact sites between plant cells and invading pathogens. To assess the role of N-glycans in receptor-mediated immune responses, we investigated the functionality of Arabidopsis receptor kinases EFR and FLS2, sensing bacterial translation elongation factor Tu (elf18) and flagellin (flg22), respectively, in N-glycosylation mutants. As revealed by binding and responses to elf18 or flg22, both receptors tolerated immature N-glycans induced by mutations in various Golgi modification steps. EFR was specifically impaired by loss-of-function mutations in STT3A, a subunit of the endoplasmic reticulum resident oligosaccharyltransferase complex. FLS2 tolerated mild underglycosylation occurring in stt3a but was sensitive to severe underglycosylation induced by tunicamycin treatment. EFR accumulation was significantly reduced when synthesized without N-glycans but to lesser extent when underglycosylated in stt3a or mutated in single amino acid positions. Interestingly, EFR N143Q lacking a single conserved N-glycosylation site from the EFR ectodomain accumulated to reduced levels and lost the ability to bind its ligand and to mediate elf18-elicited oxidative burst. However, EFR-YFP protein localization and peptide:N-glycosidase F digestion assays support that both EFR produced in stt3a and EFR N143Q in wild type cells correctly targeted to the plasma membrane via the Golgi apparatus. These results indicate that a single N-glycan plays a critical role for receptor abundance and ligand recognition during plant-pathogen interactions at the cell surface.

Cold, hyperosmolarity, and abscisic acid (ABA) signaling induce RD29A expression, which is an indicator of the plant stress adaptation response. Two nonallelic Arabidopsis thaliana (ecotype C24) T-DNA insertional mutations, cpl1 and cpl3, were identified based on hyperinduction of RD29A expression that was monitored by using the luciferase (LUC) reporter gene (RD29A::LUC) imaging system. Genetic linkage analysis and complementation data established that the recessive cpl1 and cpl3 mutations are caused by T-DNA insertions in AtCPL1 (Arabidopsis C-terminal domain phosphatase-like) and AtCPL3, respectively. Gel assays using recombinant AtCPL1 and AtCPL3 detected innate phosphatase activity like other members of the phylogenetically conserved family that dephosphorylate the C-terminal domain of RNA polymerase II (RNAP II). cpl1 mutation causes RD29A::LUC hyperexpression and transcript accumulation in response to cold, ABA, and NaCl treatments, whereas the cpl3 mutation mediates hyperresponsiveness only to ABA. Northern analysis confirmed that LUC transcript accumulation also occurs in response to these stimuli. cpl1 plants accumulate biomass more rapidly and exhibit delayed flowering relative to wild type whereas cpl3 plants grow more slowly and flower earlier than wild-type plants. Hence AtCPL1 and AtCPL3 are negative regulators of stress responsive gene transcription and modulators of growth and development. These results suggest that C-terminal domain phosphatase regulation of RNAP II phosphorylation status is a focal control point of complex processes like plant stress responses and development. AtCPL family members apparently have both unique and overlapping transcriptional regulatory functions that differentiate the signal output that determines the plant response. P lants tolerate environmental stress because of numerous physiological adaptations, which have been attributed to the function of various determinant genes (1). In Arabidopsis thaliana, transcription of RD (Responsive to Dehydration) (2) and COR (Cold Responsive) (3) genes is activated by cold or hyperosmotic stress. The plant hormone abscisic acid (ABA) activates transcription of some RD and COR genes through an interaction involving the cis element ABRE (ABA-responsive element) and basic leucine zipper transcription factors such as ABFs͞AREBs (4, 5). However, expression of some RD or COR genes is activated by low temperature or desiccation, independent of ABA, by the interaction of CBF͞DREB DNA-binding proteins with another cis element, DRE (6, 7). Overexpression of CBF͞DREB in transgenic Arabidopsis plants induces ectopic expression of RD͞COR genes and confers desiccation and cold tolerance (7,8).Recent genetic dissection of cold-, hyperosmolarity-, and ABA-induced signaling that regulates gene expression and adaptation indicates that the cascade signature is modulated by numerous positive and negative regulators (9-14). Signaling control of plant gene expression is known now to include components that function at various stages in mRNA metaboli...