Auxin Biosynthesis: Are the Indole-3-Acetic Acid and Phenylacetic Acid Biosynthesis Pathways Mirror Images?

The biosynthesis of the main auxin in plants (indole-3-acetic acid [IAA]) has been elucidated recently and is thought to involve the sequential conversion of Trp to indole-3-pyruvic acid to IAA However, the pathway leading to a less well studied auxin, phenylacetic acid (PAA), remains unclear. Here, we present evidence from metabolism experiments that PAA is synthesized from the amino acid Phe, via phenylpyruvate. In pea (Pisum sativum), the reverse reaction, phenylpyruvate to Phe, is also demonstrated. However, despite similarities between the pathways leading to IAA and PAA, evidence from mutants in pea and maize (Zea mays) indicate that IAA biosynthetic enzymes are not the main enzymes for PAA biosynthesis. Instead, we identified a putative aromatic aminotransferase (PsArAT) from pea that may function in the PAA synthesis pathway.

Seed filling in domesticated maize and rice depends on SWEET-mediated hexose transport.

Carbohydrate import into seeds directly determines seed size and must have been increased through domestication. However, evidence of the domestication of sugar translocation and the identities of seed-filling transporters have been elusive. Maize ZmSWEET4c, as opposed to its sucrose-transporting homologs, mediates transepithelial hexose transport across the basal endosperm transfer layer (BETL), the entry point of nutrients into the seed, and shows signatures indicative of selection during domestication. Mutants of both maize ZmSWEET4c and its rice ortholog OsSWEET4 are defective in seed filling, indicating that a lack of hexose transport at the BETL impairs further transfer of sugars imported from the maternal phloem. In both maize and rice, SWEET4 was likely recruited during domestication to enhance sugar import into the endosperm.

A comparative glycoproteome study of developing endosperm in the hexose-deficient miniature1 (mn1) seed mutant and its wild type Mn1 in maize.

In maize developing seeds, transfer cells are prominently located at the basal endosperm transfer layer (BETL). As the first filial cell layer, BETL is a gateway to sugars, nutrients and water from mother plant; and anchor of numerous functions such as sucrose turnover, auxin and cytokinin biosynthesis/accumulation, energy metabolism, defense response, and signaling between maternal and filial generations. Previous studies showed that basal developing endosperms of miniature1 (mn1) mutant seeds lacking the Mn1-encoded cell wall invertase II, are also deficient for hexose. Given the role of glucose as one of the key sugars in protein glycosylation and proper protein folding; we performed a comparative large scale glycoproteome profiling of total proteins of these two genotypes (mn1 mutant vs. Mn1 wild type) using 2D gel electrophoresis and glycosylation/total protein staining, followed by image analysis. Protein identification was done by LC-MS/MS. A total of 413 spots were detected; from which, 113 spots matched between the two genotypes. Of these, 45 showed >20% decrease/increase in glycosylation level and were selected for protein identification. A large number of identified proteins showed decreased glycosylation levels in mn1 developing endosperms as compared to the Mn1. Functional classification of proteins, showed mainly of post-translational modification, protein turnover, chaperone activities, carbohydrate and amino acid biosynthesis/transport, and cell wall biosynthesis. These proteins and activities were related to endoplasmic reticulum (ER) stress and unfolded protein response (UPR) as a result of the low glycolsylation levels of the mutant proteins. Overall, these results provide for the first time a global glycoproteome profile of maize BETL-enriched basal endosperm to better understand their role in seed development in maize.

Proteomic comparison of basal endosperm in maize miniature1 mutant and its wild-type Mn1.

Developing endosperm in maize seed is a major site for biosynthesis and storage of starch and proteins, and of immense economic importance for its role in food, feed and biofuel production. The basal part of endosperm performs a major role in solute, water and nutrition acquisition from mother plant to sustain these functions. The miniature1 (mn1) mutation is a loss-of-function mutation of the Mn1-encoded cell wall invertase that is entirely expressed in the basal endosperm and is essential for many of the metabolic and signaling functions associated with metabolically released hexose sugars in developing endosperm. Here we report a comparative proteomic study between Mn1 and mn1 basal endosperm to better understand basis of pleiotropic effects on many diverse traits in the mutant. Specifically, we used iTRAQ based quantitative proteomics combined with Gene Ontology (GO) and bioinformatics to understand functional basis of the proteomic information. A total of 2518 proteins were identified from soluble and cell wall associated protein (CWAP) fractions; of these 131 proteins were observed to be differentially expressed in the two genotypes. The main functional groups of proteins that were significantly different were those involved in the carbohydrate metabolic and catabolic process, and cell homeostasis. The study constitutes the first proteomic analysis of basal endosperm cell layers in relation to endosperm growth and development in maize.

Impaired auxin biosynthesis in the defective endosperm18 mutant is due to mutational loss of expression in the ZmYuc1 gene encoding endosperm-specific YUCCA1 protein in maize.

The phytohormone auxin (indole-3-acetic acid [IAA]) plays a fundamental role in vegetative and reproductive plant development. Here, we characterized a seed-specific viable maize (Zea mays) mutant, defective endosperm18 (de18) that is impaired in IAA biosynthesis. de18 endosperm showed large reductions of free IAA levels and is known to have approximately 40% less dry mass, compared with De18. Cellular analyses showed lower total cell number, smaller cell volume, and reduced level of endoreduplication in the mutant endosperm. Gene expression analyses of seed-specific tryptophan-dependent IAA pathway genes, maize Yucca1 (ZmYuc1), and two tryptophan-aminotransferase co-orthologs were performed to understand the molecular basis of the IAA deficiency in the mutant. Temporally, all three genes showed high expression coincident with high IAA levels; however, only ZmYuc1 correlated with the reduced IAA levels in the mutant throughout endosperm development. Furthermore, sequence analyses of ZmYuc1 complementary DNA and genomic clones revealed many changes specific to the mutant, including a 2-bp insertion that generated a premature stop codon and a truncated YUC1 protein of 212 amino acids, compared with the 400 amino acids in the De18. The putative, approximately 1.5-kb, Yuc1 promoter region also showed many rearrangements, including a 151-bp deletion in the mutant. Our concurrent high-density mapping and annotation studies of chromosome 10, contig 395, showed that the De18 locus was tightly linked to the gene ZmYuc1. Collectively, the data suggest that the molecular changes in the ZmYuc1 gene encoding the YUC1 protein are the causal basis of impairment in a critical step in IAA biosynthesis, essential for normal endosperm development in maize.

Pleiotropy and its dissection through a metabolic gene Miniature1 (Mn1) that encodes a cell wall invertase in developing seeds of maize.

The Mn1-encoded endosperm-specific cell wall invertase is a major determinant of sink strength of developing seeds through its control of both sink size, cell number and cell size, and sink activity via sucrose hydrolysis and release of hexoses essential for energy and signaling functions. Consequently, loss-of-function mutations of the gene lead to the mn1 seed phenotype that shows ∼70% reduction in seed mass at maturity and several pleiotropic changes. A comparative analysis of endosperm and embryo mass in the Mn1 and mn1 genotypes showed here significant reductions of both tissues in the mn1 starting with early stages of development. Clearly, embryo development was endosperm-dependent. To gain a mechanistic understanding of the changes, sugar levels were measured in both endosperm and embryo samples. Changes in the levels of all sugars tested, glc, fru, suc, and sorbitol, were mainly observed in the endosperm. Greatly reduced fru levels in the mutant led to RNA level expression analyses by q-PCR of several genes that encode sucrose and fructose metabolizing enzymes. The mn1 endosperm showed reductions in gene expression, ranging from ∼70% to 99% of the Mn1 samples, for both suc-starch and suc--energy pathways, suggesting an in vivo metabolic coordinated regulation due to the hexose-deficiency. Together, these data provide evidence of the Mn1-dependent interconnected network of several pathways as a possible basis for pleiotropic changes in seed development.

Spatial and temporal profiles of cytokinin biosynthesis and accumulation in developing caryopses of maize.

BACKGROUND AND AIMS: Cytokinins are a major group of plant hormones and are associated with various developmental processes. Developing caryopses of maize have high levels of cytokinins, but little is known about their spatial and temporal distribution. The localization and quantification of cytokinins was investigated in maize (Zea mays) caryopsis from 0 to 28 d after pollination together with the expression and localization of isopentenyltransferase ZmIPT1 involved in cytokinin biosynthesis and ZmCNGT, the gene putatively involved in N9-glucosylation. METHODS: Biochemical, cellular and molecular approaches resolved the overall cytokinin profiles, and several gene expression assays were used for two critical genes to assess cytokinin cell-specific biosynthesis and conversion to the biologically inactive form. Cytokinins were immunolocalized for the first time in maize caryopses. KEY RESULTS: During the period 0-28 d after pollination (DAP): (1) large quantities of cytokinins were detected in the maternal pedicel region relative to the filial tissues during the early stages after fertilization; (2) unpollinated ovules did not accumulate cytokinins; (3) the maternal nucellar region showed little or no cytokinin signal; (4) the highest cytokinin concentrations in filial endosperm and embryo were detected at 12 DAP, predominantly zeatin riboside and zeatin-9-glucoside, respectively; and (5) a strong cytokinin immuno-signal was detected in specific cell types in the pedicel, endosperm and embryo. CONCLUSIONS: The cytokinins of developing maize caryopsis may originate from both local syntheses as well as by transport. High levels of fertilization-dependent cytokinins in the pedicel suggest filial control on metabolism in the maternal tissue; they may also trigger developmental programmed cell death in the pedicel.

Sugar-hormone cross-talk in seed development: two redundant pathways of IAA biosynthesis are regulated differentially in the invertase-deficient miniature1 (mn1) seed mutant in maize.

The miniature1 (mn1) seed phenotype is a loss-of-function mutation at the Mn1 locus that encodes a cell wall invertase; its deficiency leads to pleiotropic changes including altered sugar levels and decreased levels of IAA throughout seed development. To understand the molecular details of such a sugar-hormone relationship, we have initiated studies on IAA biosynthesis genes in developing seeds of maize. Two tryptophan-dependent pathways of IAA biosynthesis, tryptamine (TAM) and indole-3-pyruvic acid (IPA), are of particular interest. We report on molecular isolation and characterization of an endosperm-specific ZmTARelated1 (ZmTar1) gene of the IPA branch; we have also reported recently on ZmYuc1 gene in the TAM branch. Comparative gene expression analyses here have shown that (1) the ZmTar1 transcripts were approximately 10-fold higher levels than the ZmYuc1; (2) although both genes showed the highest level of expression at 8-12 d after pollination (DAP) coincident with an early peak in IAA levels, the two showed highly divergent (antagonistic) response at 12 and 16 DAP but similar patterns at 20 and 28 DAP in the Mn1 and mn1 endosperm. The Western blot analyses for the ZmTAR1 protein, however, displayed disconcordant protein/transcript expression patterns. Overall, these data report novel observations on redundant trp-dependent pathways of auxin biosynthesis in developing seeds of maize, and suggest that homeostatic control of IAA in this important sink is highly complex and may be regulated by both sucrose metabolism and developmental signals.

Sugar levels regulate tryptophan-dependent auxin biosynthesis in developing maize kernels.

The maize (Zea mays) Miniature1 (Mn1) locus encodes the cell wall invertase INCW2, which is localized predominantly in the basal endosperm transfer layer of developing kernels and catalyzes the conversion of sucrose into glucose and fructose. Mutations in Mn1 result in pleiotropic changes, including a reduction in kernel mass and a recently reported decrease in indole-3-acetic acid (IAA) levels throughout kernel development. Here, we show that mn1-1 basal kernel regions (pedicels and basal endosperm transfer layer) accumulate higher levels of sucrose and lower levels of glucose and fructose between 8 and 28 d after pollination when compared with the wild type, whereas upper regions of mn1 accumulate similar or increased concentrations of sugars. To determine the cause of the reduction in IAA accumulation, we investigated transcript levels of several potential IAA biosynthetic enzymes. We demonstrate that reduced IAA levels most closely correspond to reduced transcript levels of ZmYUCCA (ZmYUC), a newly identified homolog of the Arabidopsis (Arabidopsis thaliana) gene YUCCA. We further demonstrate that ZmYUC catalyzes the N-hydroxylation of tryptamine and that sugar levels regulate transcript levels of ZmYUC, both in in vitro-cultured kernels and in a promoter-reporter fusion in Arabidopsis. These results indicate that developing seeds may modulate growth by altering auxin biosynthesis in response to sugar concentrations.

Short-term high temperature growth conditions during vegetative-to-reproductive phase transition irreversibly compromise cell wall invertase-mediated sucrose catalysis and microspore meiosis in grain sorghum (Sorghum bicolor).

Grain sorghum (Sorghum bicolor) crop yield is significantly compromised by high temperature stress-induced male sterility, and is attributed to reduced cell wall invertase (CWI)-mediated sucrose hydrolysis in microspores and anthers leading to altered carbohydrate metabolism and starch deficiency in pollen (Jain et al., 2007). Sorghum plants were grown under season-long ambient (30/20 degrees C day-time maximum/night-time minimum) or high temperature stress (HS, 36/26 degrees C) environments, or reciprocally transferred for 5-10 days between either temperature regimens through panicle and microspore developmental stages. Quantitative RT-PCR analyses for CWI gene SbIncw1, plasma membrane H(+)-ATPase (Mha1) and sugar transporter proteins (OsSUT3 and OsMST7 homologs in sorghum), starch deficiency and pollen sterility data are presented to confirm HS-sensitivity of pre- and post-meiotic stages of sorghum microsporogenesis. Heat stress-induced reduction in Incw transcriptional activity during microspore meiosis was irreversible despite return of optimal growth temperature conditions through further reproductive development.

A comparative study on the role of cytokinins in caryopsis development in the maize miniature1 seed mutant and its wild type.

We report here on a comparative developmental profile of plant hormone cytokinins in relation to cell size, cell number and endoreduplication in developing maize caryopsis of a cell wall invertase-deficient miniature1 (mn1) seed mutant and its wild type, Mn1, genotype. Both genotypes showed extremely high levels of total cytokinins during the very early stages of development, followed by a marked and genotype specific reduction. While the decrease of cytokinins in Mn1 was associated with their deactivation by 9-glucosylation, the absolute and the relative part of active cytokinin forms was higher in the mutant. During the exponential growth phase of endosperm between 6 d after pollination and 9 d after pollination, the mean cell doubling time, the absolute growth rate and the level of endoreduplication were similar in the two genotypes. However, the entire duration of growth was longer in Mn1 compared with mn1, resulting in a significantly higher cell number in the Mn1 endosperm. These data correlate with the previously reported peak levels of the Mn1-encoded cell wall invertase-2 (INCW2) at 12 d after pollination in the Mn1 endosperm. A model showing possible crosstalk among cytokinins, cell cycle and cell wall invertase as causal to increased cell number and sink strength of the Mn1 developing endosperm is discussed.

Miniature1-encoded cell wall invertase is essential for assembly and function of wall-in-growth in the maize endosperm transfer cell.

The miniature1 (mn1) seed phenotype in maize (Zea mays) is due to a loss-of-function mutation at the Mn1 locus that encodes a cell wall invertase (INCW2) that localizes exclusively to the basal endosperm transfer cells (BETCs) of developing seeds. A common feature of all transfer cells is the labyrinth-like wall-in-growth (WIG) that increases the plasma membrane area, thereby enhancing transport capacity in these cells. To better understand WIG formation and roles of INCW2 in the BETC development, we examined wild-type and mn1 mutant developing kernels by cryofixation and electron microscopy. In Mn1 seeds, WIGs developed uniformly in the BETC layer during 7 to 17 d after pollination, and the secretory/endocytic organelles proliferated in the BETCs. Mitochondria accumulated in the vicinity of WIGs, suggesting a functional link between them. In the mn1 BETCs, WIGs were stunted and their endoplasmic reticulum was swollen; Golgi density in the mutant BETCs was 51% of the Mn1 Golgi density. However, the polarized distribution of mitochondria was not affected. INCW2-specific immunogold particles were detected in WIGs, the endoplasmic reticulum, Golgi stacks, and the trans-Golgi network in the Mn1 BETCs, while immunogold particles were extremely rare in the mutant BETCs. Levels of WIG development in the empty pericarp4 mutant was heterogeneous among BETCs, and INCW2 immunogold particles were approximately four times more abundant in the larger WIGs than in the stunted WIGs. These results indicate that polarized secretion is activated during WIG formation and that INCW2 is required for normal development of WIGs to which INCW2 is localized.

A cellular study of teosinte Zea mays subsp. parviglumis (Poaceae) caryopsis development showing several processes conserved in maize.

The evolutionary history of maize (Zea mays subsp. mays) is of general interest because of its economic and scientific importance. Here we show that many cellular traits described previously in developing caryopses of maize are also seen in its wild progenitor teosinte (Zea mays subsp. parviglumis). These features, each with a possible role in development, include (1) an early programmed cell death in the maternal placento-chalazal (P-C) layer that may lead to increased hydrolytic conductance to the developing seed; (2) accumulation of phenolics and flavonoids in the P-C layer that may be related to antimicrobial activity; (3) formation of wall ingrowths in the basal endosperm transfer layer (BETL); (4) localization of cell wall invertase in the BETL, which is attributed to the increased transport capacity of photosynthates to the sink; and (5) endoreduplication in endosperm nuclei suggested to contribute to increased gene expression and greater sink capacity of the developing seed. In maize caryopsis, these cellular traits have been previously attributed to domestication and selection for larger seed size and vigor. Given the conservation of the entire cellular program in developing teosinte caryopses described here, we suggest that these traits evolved independently of domestication and predate human selection pressure.

Cloning and expression analyses of sucrose non-fermenting-1-related kinase 1 (SnRK1b) gene during development of sorghum and maize endosperm and its implicated role in sugar-to-starch metabolic transition.

A full-length cDNA clone, SbSnRK1b (1530 bp, GenBank accession no. EF544393), encoding a putative serine/threonine protein kinase homologue of yeast (Saccharomyces cerevisiae) SNF1, was isolated from developing endosperm of sorghum [Sorghum bicolor (L.) Moench]. Multiple sequence alignment data showed a phylogenetic affiliation of the sorghum clone with the SnRK1b group of protein kinases that are highly expressed in cereal seed endosperm. The DNA gel blot analyses indicated that SbSnRK1b gene is present as a single- or low copy number gene in sorghum. The RNA and protein gel blot analyses confirmed the expression of SbSnRK1b in developing sorghum caryopses, overlapping with the starch biosynthesis phase, 12-24 days after fertilization. In situ hybridization and immunolocalization data resolved the spatial specificity of SbSnRK1b expression in the basal endosperm transfer cell layer, the unique port of assimilate unloading in the growing sorghum seed. Expression of SbSnRK1b was also evident in the developing sorghum microspores, coincident with the onset of starch deposition phase. As in sorghum, similar spatiotemporal specificity of SnRK1b expression was observed during maize (Zea mays L.) seed development. However, discordant in situ hybridization and immunolocalization data indicated that the expression of SbSnRK1b homologue in maize is under posttranscriptional control during endosperm development.

Expression of cell wall invertase and several other genes of sugar metabolism in relation to seed development in sorghum (Sorghum bicolor).

We report expression profiles of several genes of carbohydrate metabolism, cell wall invertase (CWI) in particular, to better understand sugar transport and its utilization in developing caryopses of grain sorghum [Sorghum bicolor (L.) Moench]. Gene expression analyses for CWI using RNA gel blot and real-time quantitative PCR approaches on developing caryopses, including the glumes (maternal tissue appended to the seeds), showed expression of SbIncw (ZmIncw2 ortholog) primarily in the basal sugar unloading zone of endosperm. The expression of ZmIncw1 ortholog was significantly less abundant and restricted to the glumes. The protein and enzyme activity data corroborated the temporal transcript expression profile that showed maximal CWI protein (INCW) expression preceding the starch-filling phase of endosperm development, i.e. 6-12d-after-pollination (DAP). Protein gel blot analysis using polyclonal maize INCW1 antibodies showed a single polypeptide of 72kDa. The highest level of enzyme activity was unique to the basal part of the endosperm, in particular the basal endosperm transfer cell (BETC) layer and the maternal pedicel region that were highly enriched for the INCW protein, as seen by immunolocalization. High hexose-to-sucrose ratio in 6-12 DAP seeds, and negligible starch deposition in glumes corroborated the CWI activity data. Additionally, we report transcription profiles of several other genes related to sugar-to-starch metabolism in developing sorghum endosperm. As in maize, the INCW-mediated apoplastic cleavage of sucrose in the BETC and pedicel during the early developmental stages of caryopses is essential for the normal development of filial tissues. The unique cell-specificity of the INCW protein to both proximal and distal ends of placental sac shown here for the first time is likely to greatly increase uptakes of both hexose sugars and water through turgor sensing into developing seed. This trait is unique to sorghum among cereals and may facilitate its survival in drought environment.

Cell wall invertase-deficient miniature1 kernels have altered phytohormone levels.

The Zea mays (maize) miniature1 (Mn1) locus encodes the cell wall invertase INCW2, which is localized predominantly in the basal endosperm transfer layer (BETL) of developing kernels and catalyzes conversion of sucrose into glucose and fructose. Mutations in Mn1 result in numerous changes that include a small kernel phenotype resulting from both decreased cell size and number. To explore the pleiotropic effects of this mutation, we investigated the levels of indole-3-acetic acid (IAA), abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA) in basal regions, upper regions, and embryos of developing kernels in the inbred line W22. We measured phytohormones from 6 to 28 days after pollination (DAP) in wild type (WT) and two alleles of mn1, mn1-1 and mn1-89. IAA was the predominant hormone in kernels, with WT levels of free IAA accumulating over time to more than 2microg/g of fresh weight. Kernels of mn1-1 accumulated up to 10-fold less IAA than WT, and levels of IAA sugar conjugates were similarly reduced. Although less abundant, differences were also observed in levels of ABA, JA, and SA between WT and the mn1 alleles. SA levels were increased by as much as 10-fold in mn1-1, and mn1-89 displayed intermediate SA levels at most timepoints. These findings indicate that invertase-mediated sucrose cleavage directly or indirectly regulates the levels of key plant hormones during seed development.

Effects of season-long high temperature growth conditions on sugar-to-starch metabolism in developing microspores of grain sorghum (Sorghum bicolor L. Moench).

High temperature stress-induced male sterility is a critical problem in grain sorghum (Sorghum bicolor L. Moench) that significantly compromises crop yields. Grain sorghum plants were grown season-long under ambient (30/20 degrees C, day-time maximum/night-time minimum) and high temperature (36/26 degrees C) conditions in sunlit Soil-Plant-Atmospheric-Research (SPAR) growth chambers. We report data on the effects of high temperature on sugar levels and expression profiles of genes related to sugar-to-starch metabolism in microspore populations represented by pre- and post-meiotic "early" stages through post-mitotic "late" stages that show detectable levels of starch deposition. Microspores from high temperature stress conditions showed starch-deficiency and considerably reduced germination, translating into 27% loss in seed-set. Sugar profiles showed significant differences in hexose levels at both "early" and "late" stages at the two temperature regimes; and most notably, undetectable sucrose and approximately 50% lower starch content in "late" microspores from heat-stressed plants. Northern blot, quantitative PCR, and immunolocalization data revealed a significant reduction in the steady-state transcript abundance of SbIncw1 gene and CWI proteins in both sporophytic as well as microgametophytic tissues under high temperature conditions. Northern blot analyses also indicated greatly altered temporal expression profiles of various genes involved in sugar cleavage and utilization (SbIncw1, SbIvr2, Sh1, and Sus1), transport (Mha1 and MST1) and starch biosynthesis (Bt2, SU1, GBSS1, and UGPase) in heat-stressed plants. Collectively, these data suggest that impairment of CWI-mediated sucrose hydrolysis and subsequent lack of sucrose biosynthesis may be the most upstream molecular dysfunctions leading to altered carbohydrate metabolism and starch deficiency under elevated growth temperature conditions.

Fragments of ATP synthase mediate plant perception of insect attack.

Plants can perceive a wide range of biotic attackers and respond with targeted induced defenses. Specificity in plant non-self-recognition occurs either directly by perception of pest-derived elicitors or indirectly through resistance protein recognition of host targets that are inappropriately proteolyzed. Indirect plant perception can occur during interactions with pathogens, yet evidence for analogous events mediating the detection of insect herbivores remains elusive. Here we report indirect perception of herbivory in cowpea (Vigna unguiculata) plants attacked by fall armyworm (Spodoptera frugiperda) larvae. We isolated and identified a disulfide-bridged peptide (+ICDINGVCVDA-), termed inceptin, from S. frugiperda larval oral secretions that promotes cowpea ethylene production at 1 fmol leaf(-1) and triggers increases in the defense-related phytohormones salicylic acid and jasmonic acid. Inceptins are proteolytic fragments of chloroplastic ATP synthase gamma-subunit regulatory regions that mediate plant perception of herbivory through the induction of volatile, phenylpropanoid, and protease inhibitor defenses. Only S. frugiperda larvae that previously ingested chloroplastic ATP synthase gamma-subunit proteins and produced inceptins significantly induced cowpea defenses after herbivory. Digestive fragments of an ancient and essential plant enzyme, inceptin functions as a potent indirect signal initiating specific plant responses to insect attack.

Genetic control of cell wall invertases in developing endosperm of maize.

We show here that the total invertase activity in developing seeds of maize is due to two cell wall invertase (CWI) genes, Incw1 and Incw2 (Mn1). Our previous results have shown that loss-of-function mutations at the Mn1 locus lead to the miniature-1 (mn1) seed phenotype, marked by a loss of >70% of seed weight at maturity. The mn1 seed mutant is, however, non-lethal presumably because it retains a residual low level, approximately 1%, of the total CWI activity relative to the Mn1 endosperm throughout seed development. Evidence here shows that the residual activity in the mn1 mutant is encoded by the Incw1 gene. RNA level analyses, especially quantitative real-time PCR studies, showed significant spatial and temporal heterogeneity in the expression of the two CWI genes in the developing endosperm. The Mn1-encoded Incw2 transcripts were seen at the highest levels in the basal region (the sugar unloading zone) during the early phase of cell division and elongation in the endosperm. In contrast, the highest levels of Incw1 transcripts were seen in the storage phase in both the upper (storage cells) and the lower parts of the endosperm. Protein and enzyme level analyses, however, appeared to show a lack of concordance with the RNA level of expression in both the Mn1 and mn1 endosperms, indicating a possibility of post-transcriptional control in the expression of these two genes. Collectively, the data suggest an important role for apoplastic cleavage of sucrose throughout the duration of seed development; and, of the two isoforms, the INCW2 appears to control metabolic flux of sugar utilization in the developing endosperm.

The delayed initiation and slow elongation of fuzz-like short fibre cells in relation to altered patterns of sucrose synthase expression and plasmodesmata gating in a lintless mutant of cotton.

Cotton (Gossypium hirsutum L.) seed develops single-celled long fibres (lint) from the seed-coat epidermis at anthesis. Previous studies have shown that the initiation and rapid elongation of these fibres requires the expression of sucrose synthase (Sus) and, potentially, a transient closure of plasmodesmata. This study extends the previous work to examine the patterns of Sus expression and plasmodesmata gating in fuzz-like short fibres of a mutant that shows delayed initiation and much slower and reduced elongation of the fibre cells. Immunolocalization studies revealed delayed expression of Sus in the mutant seed-coat epidermis that correlates temporally and spatially with the initiation of the fibre cells. Anatomically, these short fibres differed from the normal lint in that their basal ends enlarged immediately after initiation, while the majority of the normal lint on wild-type seed did not show this enlargement until the end of elongation. Suppression of Sus expression in the seed-coat epidermis of the transgenic plants reduced the length of both lint and short fuzz fibres at maturity, suggesting that the growth of short fibres also requires high levels of Sus expression. Confocal imaging of the membrane-impermeant fluorescent solute carboxyfluorescein (CF) revealed no closure of plasmodesmata during the entire elongation period of short fibres from the mutant seed. These results show (i) the delayed initiation of fuzz-like short fibres from the mutant seed correlates with delayed or insufficient expression of Sus in a subset of seed-coat epidermal cells destined to become fibres and (ii) the much shortened elongation of the fibres from the mutant may be related to their inability to close plasmodesmata.