rgf1, a mutation reducing grain filling in maize through effects on basal endosperm and pedicel development

The maize cob presents an excellent opportunity to screen visually for mutations affecting assimilate partitioning in the developing kernel. We have identified a defective kernel mutant termed rgf1, reduced grain filling, with a final grain weight 30% of the wild type. In contrast with most defective endosperm mutants, rgf1 shows gene dosage-dependent expression in the endosperm. rgf1 kernels possess a small endosperm incompletely filling the papery pericarp, but embryo development is unaffected and the seeds are viable. The mutation conditions defective pedicel development and greatly reduces expression of endosperm transfer layer-specific markers. rgf1 exhibits striking morphological similarities to the mn1 mutant, but maps to a locus approximately 4 cM away from mn1 on chromosome 2 of maize. Despite reduced starch accumulation in the mutant, no obvious lesion in starch biosynthesis has been detected. Free sugar levels are unaltered in rgf1 endosperm. Rates of sugar uptake, measured over short (8 h) periods in cultured kernels, are increased in rgf1 compared to the wild type. rgf1 and wild-type kernels, excised at 5 DAP and cultured in vitro also develop differently in response to variations in sugar regime: glucose concentrations above 1% arrest placentochalazal development of rgf1 kernels, but have no effect on cultured wild-type kernels. These findings suggest that either uptake or perception of sugar(s) in endosperm cells at 5-10 DAP determines the rgf1 kernel phenotype.     

Auxin biosynthesis in maize

For the biosynthesis of the phytohormone indole-3-acetic acid (IAA), a number of tryptophan-dependent and -independent pathways have been discussed. Maize is an appropriate model system to analyze IAA biosynthesis particularly because high quantities of IAA conjugates are stored in the endosperm. This allowed precursor feeding experiments in a kernel culture system followed by retrobiosynthetic NMR analysis, which strongly suggested that tryptophan-dependent IAA synthesis is the predominant route for auxin biosynthesis in the maize kernel. Two nitrilases ZmNIT1 and ZmNIT2 are expressed in seeds. ZmNIT2 efficiently hydrolyzes indole-3-acetonitrile (IAN) to IAA and thus could be involved in auxin biosynthesis. Redundant pathways, e.g., via indole-3-acetaldehyde could imply that multiple mutants will be necessary to obtain IAA-deficient plants and to conclusively identify relevant genes for IAA biosynthesis.     

Abscisic Acid inhibition of endosperm cell division in cultured maize kernels

The response of developing maize (Zea mays L.) endosperm to elevated levels of abscisic acid (ABA) was investigated. Maize kernels and subtending cob sections were excised at 5 days after pollination (DAP) and placed in culture with or without 90 micromolar (±)-ABA in the medium. A decreased number of cells per endosperm was observed at 10 DAP (and later sampling times) in kernels cultured in medium containing ABA from 5 DAP, and in kernels transferred at 8 DAP to medium containing ABA, but not in kernels transferred at 11 DAP to medium containing ABA. The number of starch granules per endosperm was decreased in some treatments, but the reduction, when apparent, was comparable to the decreased number of endosperm cells. The effect on endosperm fresh weight was slight, transient, and appeared to be secondary to the effect on cell number. Mature endosperm dry weight was reduced when kernels were cultured continuously in medium containing ABA. Endosperm (+)-ABA content of kernels cultured in 0, 3, 10, 30, 100, or 300 micromolar (±)-ABA was measured at 10 DAP by indirect ELISA using a monoclonal antibody. Content of (+)-ABA in endosperms correlated negatively (R = −0.92) with endosperm cell number. On the basis of these studies we propose that during early kernel development, elevated levels of ABA decrease the rate of cell division in maize endosperm which, in turn, could limit the storage capacity of the kernel.     

Effect of Deseeding on the Indole-3-acetic Acid Content of Shoots and Roots of Zea mays Seedlings

We wished to determine the effect of endosperm removal on the amounts of free and esterified indole-3-acetic acid (IAA) in young Zea mays seedlings. The increases of IAA derived from endosperm and from biosynthesis, but without correction for catabolic losses, were 0.9 picomole of free IAA per shoot per hour, and 1.1 picomoles per shoot per hour of ester IAA. After deseeding, free IAA in the shoot declines by 40% following kernel removal and total (free + ester) IAA declines at a rate of about 1 picomole per shoot per hour. A slight, but insignificant increase of ester IAA occurs following endosperm removal. In the primary roots, the decreases of free IAA and total (free + ester) IAA are accelerated by seed removal. Thus, the endosperm appears to be a major source of IAA for the shoot and root.     

重穗型杂交水稻籽粒灌浆过程中强势和弱势颖花内源IAA、ABA和GA水平的动态状况

研究了重穗型杂交水稻培矮 6 4s/E3 2的灌浆过程和强、弱势颖花中内源IAA、ABA和GA1 GA3水平的动态状况。籽粒发育过程中不同内源激素水平高低依次为 :IAA >GA1 GA3>ABA。IAA和ABA水平在强势颖花中较高而GA1 GA3水平在弱势颖花中较高。 3种激素水平的变化与谷粒增重速率之间均存在正相关 ,两个最高的相关系数值分别存在于单位鲜重样本的IAA含量(ng/gFW ) 与籽粒鲜重的增重速率之间 (r =0 .82 1 8 )和单个籽粒IAA含量 (ng/grain)与籽粒干重的增重速率之间 (r =0 .8485 )。推测启动和维持籽粒灌浆过程可能需要较高的IAA水平 ;ABA可能具有促进籽粒中同化物的累积和种子成熟的作用 ;GA1 GA3可能具有保持弱势颖花活性的特殊作用     

Sucrose synthase activity in developing wheat endosperms differing in maximum weight

Past research on kernel growth in wheat (Triticum aestivum) has shown that the kernel itself largely regulates the influx of sucrose for consequent starch synthesis in the endosperm of the grain. The first step in the conversion of sucrose to starch is catalyzed by sucrose synthase (EC 2.4.13). Sucrose synthase activity was assayed in developing endosperms from kernels differing in growth rate and in maximum dry weight accumulation. From 10 to 22 days after anthesis, sucrose synthase activity per wheat endosperm remained constant with respect to time in all grains. However, kernels which had higher rates of kernel growth and which achieved greatest maximum weight had consistently and significantly higher sucrose synthase activities at any point in time than did kernels with slower rates of dry matter accumulation and lower maximum weight. In addition, larger kernels had a significantly greater amount of water in which this activity could be expressed. Although the results do not implicate sucrose synthase as the “rate limiting” enzyme in wheat kernel growth, they do emphasize the importance of sucrose synthase activity in larger or more rapidly growing kernels, as compared to smaller slower growing kernels.     

重穗型杂交水稻籽粒灌浆过程中强势和强势颖花内源IAA、ABA和GA水平的动态状况

研究了重穗型杂交水稻培矮 6 4s/E3 2的灌浆过程和强、弱势颖花中内源IAA、ABA和GA1 GA3水平的动态状况。籽粒发育过程中不同内源激素水平高低依次为 :IAA >GA1 GA3>ABA。IAA和ABA水平在强势颖花中较高而GA1 GA3水平在弱势颖花中较高。 3种激素水平的变化与谷粒增重速率之间均存在正相关 ,两个最高的相关系数值分别存在于单位鲜重样本的IAA含量(ng/gFW ) 与籽粒鲜重的增重速率之间 (r =0 .82 1 8 )和单个籽粒IAA含量 (ng/grain)与籽粒干重的增重速率之间 (r =0 .8485 )。推测启动和维持籽粒灌浆过程可能需要较高的IAA水平 ;ABA可能具有促进籽粒中同化物的累积和种子成熟的作用 ;GA1 GA3可能具有保持弱势颖花活性的特殊作用     

Biochemistry of Short-Chain Alkanes (Tissue-Specific Biosynthesis of n-Heptane in Pinus jeffreyi)

Short-chain (C7-C11) alkanes accumulate as the volatile component of oleoresin (pitch) in several pine species native to western North America. To establish the tissue most amenable for use in detailed studies of short-chain alkane biosynthesis, we examined the tissue specificity of alkane accumulation and biosynthesis in Pinus jeffreyi Grev. & Balf. Short-chain alkane accumulation was highly tissue specific in both 2-year-old saplings and mature trees; heart-wood xylem accumulated alkanes up to 7.1 mg g-1 dry weight, whereas needles and other young green tissue contained oleoresin with monoterpenoid, rather than paraffinic, volatiles. These tissue-specific differences in oleoresin composition appear to be a result of tissue-specific rates of alkane and monoterpene biosynthesis; incubation of xylem tissue with [14C]sucrose resulted in accumulation of radiolabel in alkanes but not monoterpenes, whereas incubation of foliar tissue with 14CO2 resulted in the accumulation of radiolabel in monoterpenes but not alkanes. Furthermore, incubation of xylem sections with [14C]acetate resulted in incorporation of radiolabel into alkanes at rates up to 1.7 nmol h-1 g-1 fresh weight, a rate that exceeds most biosynthetic rates reported with other plant systems for the incorporation of this basic precursor into natural products. This suggests that P. jeffreyi may provide a suitable model for elucidating the enzymology and molecular biology of short-chain alkane biosynthesis.     

Seasonal Variations in Rubber Biosynthesis, 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase,and Rubber Transferase Activities in Parthenium argentatum in the Chihuahuan Desert

The rubber content and the activities of enzymes in the polyisoprenoid pathway in Parthenium argentatum (guayule) were examined throughout the growing season in field plots in the Chihuahuan Desert. The rubber content of the plants was low in July and August and slowly increased until October. From October to December there was a rapid increase in rubber formation (per plant) from 589.0 mg to 4438.0 mg. The percentage of rubber in the plants increased from 0.7% (mg/g dry weight) in August and 1.27% in October to 5.5% in December. The rapid increase in rubber formation may result from exposing the plants to low temperatures of 5 to 7[deg]C. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) was 21.1 nmol mevalonic acid (MVA) h-1 g-1 fresh weight in the bark of the lower stems in June during seedling growth and decreased to 5.1 nmol MVA h-1g-1 fresh weight in July and 2.9 nmol MVA h-1 g-1 fresh weight in September. From October to December, the activity increased from 5.0 to 29.9 nmol MVA h-1 g-1 fresh weight. The activity of rubber transferase was 65.5 nmol isopentenyl pyrophosphate (IPP) h-1 g-1fresh weight in the bark in September and increased to 357.5 nmol IPP h-1 g-1 fresh weight in December. The rapid increase in the activities of HMGR and rubber transferase coincided with the rapid increase in rubber formation. The activities of MVA kinase and IPP isomerase did not significantly increase in the fall and winter. A tomato HMGR-1 cDNA probe containing a highly conserved C-terminal region of HMGR genes hybridized at low stringency with several bands on blots of HindIII-digested genomic DNA from guayule. In northern blots with the HMGR-1 cDNA probe at low stringency, HMGR mRNA was high in June and November, corresponding to periods of high HMGR activity during seedling growth and rapid increase in rubber formation. The seasonal variations in rubber formation and HMGR mRNA, HMGR activity, and rubber transferase activity may be due to low temperature stimulation in the fall and winter months.     

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.     

Distribution of enzyme activities within the developing maize (Zea mays) kernel in relation to starch, oil and protein accumulation

The association of enzyme activities in developing kernels with specific storage product accumulation at maturity was analyzed in different parts of Zea mays inbred OH43 kernels. Maize kernels were harvested at 20 and 55 days post-pollination and dissected into basal region, pericarp, embryo, lower endosperm, middle endosperm and upper endosperm. Mature (55 days pos(-pollination) kernel parts were analyzed for starch, total protein, zein and oil content. Immature (20 days post-pollination) kernel parts were assayed for activities of 15 enzymes of sugar and amino acid metabolism. Statistical analyses of the data suggested that glucokinase (EC 2.7.1.2) fructokinase (EC 2.7.1.4) and phosphofructokinase (EC 2.7.1.1 11) activities were primarily associated with oil accumulation, whereas ADP'-glueose pyrophosphorylasc (EC 2.7.7.27) and sucrose synthase (EC 2.4.1.13) activities were associated with starch accumulation. The results suggest that oil biosynthesis utilizes inveitase-mediated sucrose degradation in a pathway not requiring pyrophosphatc. whereas starch biosynthesis utilizes a sucrose synthase-mediated pathway of sucrose degradation in a pathway requiring pyrophosphatc. Additional groups of enzyme activities were associated with each oilier but not with any specific storage product and appeared to be associated with general metabolic activity.     

Enzyme activities of starch and sucrose pathways and growth of apical and Basal maize kernels

Apical kernels of maize (Zea mays L.) ears have smaller size and lower growth rates than basal kernels. To improve our understanding of this difference, the developmental patterns of starch-synthesis-pathway enzyme activities and accumulation of sugars and starch was determined in apical- and basal-kernel endosperm of greenhouse-grown maize (cultivar Cornell 175) plants. Plants were synchronously pollinated, kernels were sampled from apical and basal ear positions throughout kernel development, and enzyme activities were measured in crude preparations. Several factors were correlated with the higher dry matter accumulation rate and larger mature kernel size of basal-kernel endosperm. During the period of cell expansion (7 to 19 days after pollination), the activity of insoluble (acid) invertase and sucose concentration in endosperm of basal kernels exceeded that in apical kernels. Soluble (alkaline) invertase was also high during this stage but was the same in endosperm of basal and apical kernels, while glucose concentration was higher in apical-kernel endosperm. During the period of maximal starch synthesis, the activities of sucrose synthase, ADP-Glc-pyrophosphorylase, and insoluble (granule-bound) ADP-Glc-starch synthase were higher in endosperm of basal than apical kernels. Soluble ADP-Glc-starch synthase, which was maximal during the early stage before starch accumulated, was the same in endosperm from apical and basal kernels. It appeared that differences in metabolic potential between apical and basal kernels were established at an early stage in kernel development.     

Characterization of auxin conjugates in Arabidopsis. Low steady-state levels of indole-3-acetyl-aspartate, indole-3-acetyl-glutamate, and indole-3-acetyl-glucose

Amide-linked indole-3-acetic acid (IAA) conjugates constitute approximately 90% of the IAA pool in the dicot Arabidopsis, whereas ester-linked conjugates and free IAA account for approximately 10% and 1%, respectively when whole seedlings are measured. We show here that IAA-aspartate Asp, IAA-glutamate (Glu), and IAA-glucose (Glc) are present at low levels in Arabidopsis. Nine-day-old wild-type Arabidopsis seedlings yielded 17.4 +/- 4.6 ng g(-1) fresh weight IAA-Asp and 3.5 +/- 1.6 ng g(-1) fresh weight IAA-Glu, and IAA-Glc was present at 7 to 17 ng g(-1) fresh weight in 12-d-old wild-type seedlings. Total IAA content in 9-d-old Arabidopsis seedlings was 1, 200 +/- 178 ng g(-1) fresh weight, so these three IAA conjugates together made up only 3% of the conjugate pool throughout the whole plant. We detected less than wild-type levels of IAA-Asp and IAA-Glu (7.8 +/- 0.4 ng g(-1) fresh weight and 1.8 +/- 0.3 ng g(-1) fresh weight, respectively) in an Arabidopsis mutant that accumulates conjugated IAA. Our results are consistent with IAA-Asp, IAA-Glu, and IAA-Glc being either minor, transient, or specifically localized IAA metabolites under normal growth conditions and bring into question the physiological relevance of IAA-Asp accumulation in response to high concentrations of exogenous IAA.     

Central metabolic fluxes in the endosperm of developing maize seeds and their implications for metabolic engineering

1?C labeling experiments performed with kernel cultures showed that developing maize endosperm is more efficient than other non-photosynthetic tissues such as sunflower and maize embryos at converting maternally supplied substrates into biomass. To characterize the metabolic fluxes in endosperm, maize kernels were labeled to isotopic steady state using 13C-labeled glucose. The resultant labeling in free metabolites and biomass was analyzed by NMR and GC-MS. After taking into account the labeling of substrates supplied by the metabolically active cob, the fluxes through central metabolism were quantified by computer-aided modeling. The flux map indicates that 51-69% of the ATP produced is used for biomass synthesis and up to 47% is expended in substrate cycling. These findings point to potential engineering targets for improving yield and increasing oil contents by, respectively, reducing substrate cycling and increasing the commitment of plastidic carbon into fatty acid synthesis at the level of pyruvate kinase.     

Regulation of programmed cell death in maize endosperm by abscisic acid

Cereal endosperm undergoes programmed cell death (PCD) during its development, a process that is controlled, in part, by ethylene. Whether other hormones influence endosperm PCD has not been investigated. Abscisic acid (ABA) plays an essential role during late seed development that enables an embryo to survive desiccation. To examine whether ABA is also involved in regulating the onset of PCD during endosperm development, we have used genetic and biochemical means to disrupt ABA biosynthesis or perception during maize kernel development. The onset and progression of cell death, as determined by viability staining and the appearance of internucleosomal DNA fragmentation, was accelerated in developing endosperm of ABA-insensitive vp1 and ABA-deficient vp9 mutants. Ethylene was synthesized in vp1 and vp9 mutant kernels at levels that were 2–4-fold higher than in wild-type kernels. Moreover, the increase and timing of ethylene production correlated with the premature onset and accelerated progression of internucleosomal fragmentation in these mutants. Treatment of developing wild-type endosperm with fluridone, an inhibitor of ABA biosynthesis, recapitulated the increase in ethylene production and accelerated execution of the PCD program that was observed in the ABA mutant kernels. These data suggest that a balance between ABA and ethylene establishes the appropriate onset and progression of programmed cell death during maize endosperm development.     

Urea synthesis from ammonia in periportal and pericentral regions of the liver lobule. Effect of oxygen

Rates of urea synthesis were determined in periportal and pericentral regions of the liver lobule in perfused liver from fed, phenobarbital-treated rats by measuring the extra O2 consumed upon infusion of NH4Cl with miniature O2 electrodes and from decreases in NADPH fluorescence detected with micro-light-guides. Urea synthesis by the perfused rat liver supplemented with lactate (5 mM), ornithine (2 mM) and methionine sulfoximine (0.15 mM), an inhibitor of glutamine synthetase, was stimulated by stepwise infusion of NH4Cl at doses ranging from 0.24 mM to 3.0 mM. A good correlation (r = 0.92) between decreases in NADPH fluorescence and urea production was observed when the NH4Cl concentration was increased. Sublobular rates of O2 uptake were determined by placing miniature oxygen electrodes on periportal or pericentral regions of the lobule on the liver surface, stopping the flow and measuring decreases in oxygen tension. From such measurements local rates of O2 uptake were calculated in the presence and absence of NH4Cl and local rates of urea synthesis were calculated from the extra O2 consumed in the presence of NH4Cl and the stoichiometry between O2 uptake and urea formation. Rates of urea synthesis were also estimated from the fractional decrease in NADPH fluorescence, caused by NH4Cl infusion in each region, measured with micro-light-guides and the rate of urea synthesis by the whole organ. When perfusion was in the anterograde direction, maximal rates of urea synthesis, calculated from changes in fluorescence, were 177 +/- 31 mumol g-1 h-1 and 61 +/- 24 mumol g-1 h-1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, however, rates were 76 +/- 23 mumol g-1 h-1 in periportal areas and 152 +/- 19 mumol g-1 h-1 in pericentral regions. During perfusion in the anterograde direction, urea synthesis, calculated by changes in O2 uptake, was 307 +/- 76 mumol g-1 h-1 and 72 +/- 34 mumol g-1 h-1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, urea was synthesized at rates of 54 +/- 17 mumol g-1 h-1 and 387 +/- 99 mumol g-1 h-1 in periportal and pericentral regions, respectively. Thus, maximal rates of urea synthesis were dependent upon the direction of perfusion. In addition, rates of urea synthesis were elevated dramatically in periportal regions when the flow rate per gram liver was increased (e.g. 307 versus 177 mumol g-1 h-1).(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential inhibition of indole-3-acetic acid and tryptophan biosynthesis by indole analogues. I. Tryptophan dependent IAA biosynthesis

Maize liquid endosperm extracts contain the enzymes necessary for all of the steps of the plant IAA biosynthetic pathway from tryptophan, and provide a means to assay the pathway in vitro. We have analyzed the reactions in the presence of a series of indole and indole-like analogues in order to evaluate the potential of these compounds to act as inhibitors of IAA biosynthesis. Such inhibitors will be useful to investigate the tryptophan to IAA pathway, to determine the precursors and intermediates involved, and to select for mutants in this process. A number of such compounds were tested using in vitro enzyme assays for both the tryptophan dependent IAA biosynthesis pathway and for tryptophan synthase activity. Some compounds showed strong inhibition of IAA biosynthesis while having only a slight effect on the reaction rate of tryptophan synthase . These results: (1) show that IAA biosynthesis can be selectively inhibited relative to tryptophan biosynthesis; (2) suggest potential ways to screen for IAA biosynthetic pathway mutations in plants; and (3) provide additional tools for studies of IAA biosynthesis in plants.     

Abscisic acid catabolism in maize kernels in response to water deficit at early endosperm development

To further our understanding of the greater susceptibility of apical kernels in maize inflorescences to water stress, abscisic acid (ABA) catabolism activity was evaluated in developing kernels with chirally separated (+)-[(3)H]ABA. The predominant pathway of ABA catabolism was via 8'-hydroxylase to form phaseic acid, while conjugation to glucose was minor. In response to water deficit imposed on whole plants during kernel development, ABA accumulated to higher concentrations in apical than basal kernels, while both returned to control levels after rewatering. ABA catabolism activity per gram fresh weight increased about three-fold in response to water stress, but was about the same in apical and basal kernels on a fresh weight basis. ABA catabolism activity was three to four-fold higher in placenta than endosperm, and activity was higher in apical than basal kernels. In vitro incubation tests indicated that glucose did not affect ABA catabolism. We conclude that placenta tissue plays an important role in ABA catabolism, and together with ABA influx and compartmentation, determine the rate of ABA transport into endosperms.