Occurrence of Pseudomonas cichorii causing midrib rot of lettuce under hydroponic cultivation in Chile.

In Chile, lettuce is the vegetable that has increased in cultivated area the most in recent years, reaching 8,309 ha. The Coquimbo Region contributes the most to this growth in production with 3,284 ha in 2022 (ODEPA 2023). Most lettuce is grown under open field conditions, but there is significant production in greenhouses and an increase in hydroponic production systems (INIA 2017). During April to June 2021 and 2022 in the Coquimbo Region, butterhead-type lettuce seedlings (Lactuca sativa) cv. Neil, cultivated under a hydroponic system, showed severe brown to black lesions in the leaves and midrib (Figure S1). To determine the etiology of this problem, samples of diseased plants were taken. Pieces of symptomatic tissue were macerated, and the extract was spread on nutrient agar (NA) and on King's B medium (KB) and incubated at 23°C for 48 h. The bacterial colonies observed were predominantly circular, creamy-white in color with irregular margins and fluorescent in KB medium. Isolates were gram-negative strictly aerobic. LOPAT test (Lelliot et al. 1966) results of two selected isolates were: levan production (-), oxidase reaction (+), potato soft rot (-), arginine dihydrolase production (-), and tobacco hypersensitivity (+), which corresponds to the profile of Pseudomonas cichorii. Molecular identification was performed through amplification and sequencing of the 16S rRNA (GenBank Accessions No. OR540674 to OR540675), gyrB and rpoD genes (Hwang et al. 2005; Sarkar and Guttmann 2004) (GenBank Accessions No. OR558279 to OR558282). BLAST analysis of the 16S rRNA gene of the isolates resulted in a match with a 99.86% identity with P. cichorii type strain ATCC 10857 (NR_112070.1). BLAST analysis of gyrB and rpoD resulted in a match with a 100% (630/630 bp) and >99% (546/550 bp) identity respectively, with strains of P. cichorii. Five six-month-old lettuce plants cv. Desert Storm were pricked in the midrib with a toothpick smeared with a fresh colony grown on KB medium. Seven days after inoculation, the plants showed dark brown, watery lesions, characteristic of damage caused by P. cichorii (Figure S1). Bacteria were isolated again from the inoculated plants and were identified as P. cichorii using LOPAT and molecular identification techniques. Midrib rot caused by P. cichorii was reported as an emerging disease of greenhouse-grown lettuce by Cottyn et al. (2009). In Chile, P. cichorii was previously described affecting nectarine fruits (Pinto de Torres and Carreño Ibañez 1983) and reported as a pathogen of lettuce among others horticultural crops by Servicio Agrícola y Ganadero of the Government of Chile (Acuña 2008), but this is the first report of P. cichorii affecting hydroponic lettuce plants in Chile. These results will be the basis of future studies to evaluate the origin of the infection, the potential dissemination, and the implementation of disease management to avoid the damage caused by this bacterium in hydroponic systems in this crop of growing importance in Chile.

Rewired phenolic metabolism and improved saccharification efficiency of a Zea mays cinnamyl alcohol dehydrogenase 2 (zmcad2) mutant.

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose-to-glucose conversion of maize stover. Here, we analyzed a field-grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD-deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose-to-glucose conversion efficiency was determined after 10 different thermochemical pre-treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre-treatment. However, the relative increase in glucose yields after alkaline pre-treatment was not higher than the relative increase when no pre-treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p-coumarate levels in the cell wall. Taken together, our results reveal how phenolic metabolism is affected in CAD-deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar-platform biorefineries.

Visualising the effect of freezing on the vascular system of wheat in three dimensions by in-block imaging of dye-infiltrated plants.

Infrared thermography has shown after roots of grasses freeze, ice spreads into the crown and then acropetally into leaves initially through vascular bundles. Leaves freeze singly with the oldest leaves freezing first and the youngest freezing later. Visualising the vascular system in its native 3-dimensional state will help in the understanding of this freezing process. A 2 cm section of the crown that had been infiltrated with aniline blue was embedded in paraffin and sectioned with a microtome. A photograph of the surface of the tissue in the paraffin block was taken after the microtome blade removed each 20 µm section. Two hundred to 300 images were imported into Adobe After Effects and a 3D volume of the region infiltrated by aniline blue dye was constructed. The reconstruction revealed that roots fed into what is functionally a region inside the crown that could act as a reservoir from which all the leaves are able to draw water. When a single root was fed dye solution, the entire region filled with dye and the vascular bundles of every leaf took up the dye; this indicated that the vascular system of roots was not paired with individual leaves. Fluorescence microscopy suggested the edge of the reservoir might be composed of phenolic compounds. When plants were frozen, the edges of the reservoir became leaky and dye solution spread into the mesophyll outside the reservoir. The significance of this change with regard to freezing tolerance is not known at this time. Thermal cameras that allow visualisation of water freezing in plants have shown that in crops like wheat, oats and barley, ice forms first at the bottom of the plant and then moves upwards into leaves through water conducting channels. Leaves freeze one at a time with the oldest leaves freezing first and then younger ones further up the stem freeze later. To better understand why plants freeze like this, we reconstructed a 3-dimensional view of the water conducting channels. After placing the roots of a wheat plant in a blue dye and allowing it to pull the dye upwards into leaves, we took a part of the stem just above the roots and embedded it in paraffin. We used a microtome to slice a thin layer of the paraffin containing the plant and then photographed the surface after each layer was removed. After taking about 300 images, we used Adobe After Effects software to re-construct the plant with the water conducting system in three dimensions. The 3D reconstruction showed that roots fed into a roughly spherical area at the bottom of the stem that could act as a kind of tank or reservoir from which the leaves pull up water. When we put just one root in dye, the entire reservoir filled up and the water conducting channels in every leaf took up the dye. This indicates that the water channels in roots were not directly connected to specific leaves as we had thought. When plants were frozen, the dye leaked out of the reservoir and spread into cells outside. Research is continuing to understand the significance of this change during freezing. It is possible that information about this effect can be used to help breeders develop more winter-hardy crop plants.

Effects of season, variety type, and trait on dry matter yield, nutrient composition, and predicted intake and milk yield of whole-plant sorghum forage.

Sorghum forage is an important alternative to high-quality forage in regions where climatic and soil conditions are less desirable for corn production for silage and producing comparable nutritive value is challenging. The objective of this experiment was to assess the effects of season (spring vs. summer), sorghum variety type (forage sorghum vs. sorghum-sudangrass), and trait [brown midrib (BMR) vs. non-BMR] on dry matter (DM) yield, nutrient composition, and predicted intake and milk yield of whole-plant sorghum forage grown in Florida from 2008 to 2019. Whole-plant sorghum forage was harvested at a targeted 32% of DM, and each year, spring (April) and summer (July) trials were established. A total of 300 forage sorghum and 137 sorghum-sudangrass hybrids were tested for a total of 437 hybrids, of which 199 hybrids contained the BMR trait and 238 were non-BMR. An interaction between season and sorghum variety type was observed for DM yield. Dry matter yield was greater for the spring season than the summer season, with sorghum-sudangrass outperforming forage sorghum only during the spring season. In addition, BMR hybrids had a lower DM yield than non-BMR hybrids, regardless of season and variety type. An interaction between season and trait was observed for predicted neutral detergent fiber digestibility after 30 h of incubation in rumen fluid (NDFD30h). Predicted NDFD30h was greater for BMR sorghum in comparison to non-BMR sorghum, but BMR sorghum had slightly greater predicted NDFD30h when grown in the spring than summer, whereas no seasonal differences were found for predicted NDFD30h across non-BMR sorghum. An interaction between season, variety type, and trait was observed for predicted dry matter intake at 45 (DMI45), 55 (DMI55), and 65 (DMI65) kg of milk/d. Predicted DMI45 and DMI55 were greater for spring BMR forage sorghum than for spring and summer non-BMR sorghum-sudangrass and were greater for spring BMR forage sorghum than for summer BMR sorghum-sudangrass. Predicted DMI65 was greater for BMR forage sorghum in comparison to all non-BMR hybrids in the spring. Additionally, spring BMR forage sorghum was greater than summer sorghum-sudangrass regardless of trait. An interaction between season and sorghum variety type was observed for milk yield per megagram of forage. Milk yield per megagram of forage was greatest for spring forage sorghum. Sorghum variety type and trait selection are crucial to minimize differences in forage nutritive value of sorghum forage between seasons and improve the performance of high-producing dairy cows.

Loss of COMT activity reduces lateral root formation and alters the response to water limitation in sorghum brown midrib (bmr) 12 mutant.

Lignin is a key target for modifying lignocellulosic biomass for efficient biofuel production. Brown midrib 12 (bmr12) encodes the sorghum caffeic acid O-methyltransferase (COMT) and is one of the key enzymes in monolignol biosynthesis. Loss of function mutations in COMT reduces syringyl (S) lignin subunits and improves biofuel conversion rate. Although lignin plays an important role in maintaining cell wall integrity of xylem vessels, physiological and molecular consequences due to loss of COMT on root growth and adaptation to water deficit remain unexplored. We addressed this gap by evaluating the root morphology, anatomy and transcriptome of bmr12 mutant. The mutant had reduced lateral root density (LRD) and altered root anatomy and response to water limitation. The wild-type exhibits similar phenotypes under water stress, suggesting that bmr12 may be in a water deficit responsive state even in well-watered conditions. bmr12 had increased transcript abundance of genes involved in (a)biotic stress response, gibberellic acid (GA) biosynthesis and signaling. We show that bmr12 is more sensitive to exogenous GA application and present evidence for the role of GA in regulating reduced LRD in bmr12. These findings elucidate the phenotypic and molecular consequences of COMT deficiency under optimal and water stress environments in grasses.

Influence of fiber degradability of corn silage in diets with lower and higher fiber content on lactational performance, nutrient digestibility, and ruminal characteristics in lactating Holstein cows.

The effect of neutral detergent fiber (NDF) degradability of corn silage in diets containing lower and higher NDF concentrations on lactational performance, nutrient digestibility, and ruminal characteristics in lactating Holstein cows was measured. Eight ruminally cannulated Holstein cows averaging 91 ± 4 (standard error) days in milk were used in a replicated 4 × 4 Latin square design with 21-d periods (7-d collection periods). Dietary treatments were formulated to contain either conventional (CON; 48.6% 24-h NDF degradability; NDFD) or brown midrib-3 (BM3; 61.1% 24-h NDFD) corn silage and either lower NDF (LNDF) or higher NDF (HNDF) concentration (32.0 and 35.8% of ration dry matter, DM) by adjusting the dietary forage content (52 and 67% forage, DM basis). The dietary treatments were (1) CON-LNDF, (2) CON-HNDF, (3) BM3-LNDF, and (4) BM3-HNDF. Data were analyzed as a factorial arrangement of diets within a replicated Latin square design with the MIXED procedure of SAS (SAS Institute Inc., Cary, NC) with fixed effects of NDFD, NDF, NDFD × NDF, period(square), and square. Cow within square was the random effect. Time and its interactions with NDFD and NDF were included in the model when appropriate. An interaction between NDFD and NDF content resulted in the HNDF diet decreasing dry matter intake (DMI) with CON corn silage but not with BM3 silage. Cows fed the BM3 corn silage had higher DMI than cows fed the CON corn silage, whereas cows fed the HNDF diet consumed less DM than cows fed the LNDF diet. Cows fed the BM3 diets had greater energy-corrected milk yield, higher milk true protein content, and lower milk urea nitrogen concentration than cows fed CON diets. Additionally, cows fed the BM3 diets had greater total-tract digestibility of organic matter and NDF than cows fed the CON diets. Compared with CON diets, the BMR diets accelerated ruminal NDF turnover. When incorporated into higher NDF diets, corn silage with greater in vitro 24-h NDFD and lower undegradable NDF at 240 h of in vitro fermentation (uNDF240) allowed for greater DMI intake than CON. In contrast, for lower NDF diets, NDFD of corn silage did not affect DMI, which suggests that a threshold level of inclusion of higher NDFD corn silage is necessary to observe enhanced lactational performance. Results suggest that there is a maximum gut fill of dietary uNDF240 and that higher NDFD corn silage can be fed at greater dietary concentrations.

Overexpression of ferulate 5-hydroxylase increases syringyl units in Sorghum bicolor.

Ferulate 5-hydroxylase (F5H) of the monolignol pathway catalyzes the hydroxylation of coniferyl alcohol, coniferaldehyde and ferulic acid to produce 5-hydroxyconiferyl moieties, which lead to the formation of sinapic acid and syringyl (S) lignin monomers. In contrast, guaiacyl (G) lignin, the other major type of lignin monomer, is derived from polymerization of coniferyl alcohol. In this study, the effects of manipulating S-lignin biosynthesis in sorghum (Sorghum bicolor) were evaluated. Overexpression of sorghum F5H (SbF5H), under the control of the CaMV 35S promoter, increased both S-lignin levels and the ratio of S/G lignin, while plant growth and development remained relatively unaffected. Maüle staining of stalk and leaf midrib sections from SbF5H overexpression lines indicated that the lignin composition was altered. Ectopic expression of SbF5H did not affect the gene expression of other monolignol pathway genes. In addition, brown midrib 12-ref (bmr12-ref), a nonsense mutation in the sorghum caffeic acid O-methyltransferase (COMT) was combined with 35S::SbF5H through cross-pollination to examine effects on lignin synthesis. The stover composition from bmr12 35S::SbF5H plants more closely resembled bmr12 stover than 35S::SbF5H or wild-type (WT) stover; S-lignin and total lignin concentrations were decreased relative to WT or 35S::SbF5H. Likewise, expression of upstream monolignol biosynthetic genes was increased in both bmr12 and bmr12 35S::SbF5H relative to WT or 35S::SbF5H. Overall, these results indicated that overexpression of SbF5H did not compensate for the loss of COMT activity. KEY MESSAGE: Overexpression of F5H in sorghum increases S-lignin without increasing total lignin content or affecting plant growth, but it cannot compensate for the loss of COMT activity in monolignol synthesis.

Graduate Student Literature Review: Current perspectives on whole-plant sorghum silage production and utilization by lactating dairy cows.

The purpose of this literature review is to evaluate current research into and understanding of whole-plant sorghum silage production and the effect of feeding whole-plant sorghum silage on lactation performance of dairy cows. Sorghum's drought tolerance, water efficiency, and low cost of production make it an intriguing crop in areas where whole-plant corn silage production may be limited. Currently, urban land encroachment and reduced water availability have increased social and economic pressures on farms to improve crop production efficiency. As these challenges become more prevalent, greater reliance on sorghum can be expected because of its ability to produce high dry matter yields while maintaining nutritive value, even under less-than-ideal growing conditions. Moreover, whole-plant sorghum silage provides both physically effective fiber and energy through fiber and grain fractions. Advancements in sorghum genetics and mechanical processing have the potential to alleviate common challenges associated with whole-plant sorghum silage supplementation, such as increased neutral detergent fiber and decreased neutral detergent fiber digestibility, starch concentration, and starch digestibility. These nutritive challenges must be overcome for whole-plant sorghum silage to be a viable alternative to whole-plant corn silage.

RNAi-suppression of barley caffeic acid O-methyltransferase modifies lignin despite redundancy in the gene family.

Caffeic acid O-methyltransferase (COMT), the lignin biosynthesis gene modified in many brown-midrib high-digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl-to-guaiacyl ratio was reduced by ~50%, the 5-hydroxyguaiacyl (5-OH-G) unit incorporated into lignin at 10--15-fold higher levels than normal, and the amount of p-coumaric acid ester-linked to cell walls was reduced by ~50%. No brown-midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown-midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.

Axial anatomy of the leaf midrib provides new insights into the hydraulic architecture and cavitation patterns of Acer pseudoplatanus leaves.

The structure of leaf veins is typically described by a hierarchical scheme (e.g. midrib, 1st order, 2nd order), which is used to predict variation in conduit diameter from one order to another whilst overlooking possible variation within the same order. We examined whether xylem conduit diameter changes within the same vein order, with resulting consequences for resistance to embolism. We measured the hydraulic diameter (Dh), and number of vessels (VN) along the midrib and petioles of leaves of Acer pseudoplatanus, and estimated the leaf area supplied (Aleaf-sup) at different points of the midrib and how variation in anatomical traits affected embolism resistance. We found that Dh scales with distance from the midrib tip (path length, L) with a power of 0.42, and that VN scales with Aleaf-sup with a power of 0.66. Total conductive area scales isometrically with Aleaf-sup. Embolism events along the midrib occurred first in the basipetal part and then at the leaf tip where vessels are narrower. The distance from the midrib tip is a good predictor of the variation in vessel diameter along the 1st order veins in A. pseudoplatanus leaves and this anatomical pattern seems to have an effect on hydraulic integrity since wider vessels at the leaf base embolize first.

Methane emissions of stored manure from dairy cows fed conventional or brown midrib corn silage.

The objective of this study was to examine the effects of feeding conventional corn silage (CCS) or brown midrib corn silage (BMCS) to dairy cows on CH4 emissions from stored manure. Eight lactating cows were fed (ad libitum) a total mixed ration (forage:concentrate ratio 65:35; dry matter basis) containing 59% (dry matter basis) of either CCS or BMCS. Feces and urine were collected from each cow and mixed with residual sludge obtained from a manure storage structure. Manure was incubated for 17 wk at 20°C under anaerobic conditions (O2-free N2) in 500-mL glass bottles. Methane emissions and changes in chemical composition of the manure were monitored during the incubation period. The total amount of feces and urine excreted was higher for cows fed BMCS than for cows fed CCS [8.6 vs. 6.5 kg/d of volatile solids (VS)]. Manure from cows fed BMCS emitted more CH4 than manure from cows fed CCS (173 vs. 146 L/kg of VS) throughout the incubation period. Similarly, VS and neutral detergent fiber losses throughout incubation were higher for manure from cows fed BMCS versus cows fed CCS (37.6 vs. 30.6% and 46.2 vs. 31.2%, respectively). Manure NH3 concentration (79% of total manure N) was not affected by corn silage cultivar. Results of this study show that using a more digestible corn silage cultivar (BMCS vs. CCS) may increase the contribution of manure to CH4 emissions, and may offset gain achieved by reducing enteric CH4 emissions.

Influence of a novel bm3 corn silage hybrid with floury kernel genetics on lactational performance and feed efficiency of Holstein cows.

Dry matter intake, lactation performance, and chewing behavior of multiparous Holstein cows (n = 15) fed diets containing a novel bm3 corn silage hybrid with floury kernel genetics were compared with cows fed diets containing commercially available conventional and bm3 hybrids using a replicated 3 × 3 Latin square design with 28-d periods. Cows were housed in tiestalls, milked 3 times/d, and fed a total mixed ration containing 49.0% (dry matter basis) of (1) a conventional corn silage hybrid (CONV); (2) a brown midrib bm3 hybrid (BMR); or (3) a bm3 hybrid with floury kernel genetics (BMRFL). All diets contained 6.3% hay crop silage and 44.7% concentrate. Dietary nutrient composition averaged 32.7% neutral detergent fiber (NDF) and 26.3 starch (% of dry matter). Data were analyzed by ANOVA using the MIXED procedure in SAS (SAS Institute Inc., Cary, NC). The dry matter intake was greater for cows fed BMR (28.0 kg/d) compared with CONV (26.8 kg/d), whereas dry matter intake for cows fed BMRFL was intermediate (27.6 kg/d). Energy-corrected milk (ECM) yield was greater for cows fed BMR (50.3 kg/d) and BMRFL (51.8 kg/d) compared with CONV (47.2 kg/d). Milk fat yield was higher for cows fed BMRFL (1.87 kg/d) compared with CONV (1.74 kg/d) and BMR (1.80 kg/d). Milk protein yield was greater for cows fed BMR (1.49 kg/d) and BMRFL (1.54 kg/d) compared with CONV (1.36 kg/d). Milk urea-N was reduced for cows fed BMR (11.61 mg/dL) and BMRFL (11.16 mg/dL) compared with CONV (13.60 mg/dL). Feed efficiency (ECM/dry matter intake) was higher for cows fed BMRFL (1.87) compared with CONV (1.76) and BMR (1.79). Milk N efficiency was greatest for cows fed BMRFL (40.4%) followed by BMR (38.1%) and finally CONV (35.3%). Cows fed CONV chewed 5 min more per kilograms of NDF consumed than cows fed either of the BMR hybrids. No differences were observed among diets in apparent total-tract digestibility of NDF (58.1%) or starch (99.3%). Overall lactational performance was enhanced for cows fed diets containing both BMR and BMRFL hybrids versus CONV. In addition, feeding the BMRFL corn silage improved efficiency of component-corrected milk production and milk N efficiency compared with the CONV and BMR silages.

Transcriptional Profile Corroborates that bml Mutant Plays likely Role in Premature Leaf Senescence of Rice (Oryza sativa L.).

Leaf senescence is the last period of leaf growth and a dynamic procedure associated with its death. The adaptability of the plants to changing environments occurs thanks to leaf senescence. Hence, transcriptional profiling is important to figure out the exact mechanisms of inducing leaf senescence in a particular crop, such as rice. From this perspective, leaf samples of two different rice genotypes, the brown midrib leaf (bml) mutant and its wild type (WT) were sampled for transcriptional profiling to identify differentially-expressed genes (DEGs). We identified 2670 DEGs, among which 1657 genes were up- and 1013 genes were down-regulated. These DEGs were enriched in binding and catalytic activity, followed by the single organism process and metabolic process through gene ontology (GO), while the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the DEGs were related to the plant hormone signal transduction and photosynthetic pathway enrichment. The expression pattern and the clustering of DEGs revealed that the WRKY and NAC family, as well as zinc finger transcription factors, had greater effects on early-senescence of leaf compared to other transcription factors. These findings will help to elucidate the precise functional role of bml rice mutant in the early-leaf senescence.

Methane production, ruminal fermentation characteristics, nutrient digestibility, nitrogen excretion, and milk production of dairy cows fed conventional or brown midrib corn silage.

The objective of this study was to examine the effect of replacing conventional corn silage (CCS) with brown midrib corn silage (BMCS) in dairy cow diets on enteric CH4 emission, nutrient intake, digestibility, ruminal fermentation characteristics, milk production, and N excretion. Sixteen rumen-cannulated lactating cows used in a crossover design (35-d periods) were fed (ad libitum) a total mixed ration (forage:concentrate ratio = 65:35, dry matter basis) based (59% dry matter) on either CCS or BMCS. Dry matter intake and milk yield increased when cows were fed BMCS instead of CCS. Of the milk components, only milk fat content slightly decreased when cows were fed the BMCS-based diet compared with when fed the CCS-based diet (3.81 vs. 3.92%). Compared with CCS, feeding BMCS to cows increased yields of milk protein and milk fat. Ruminal pH, protozoa numbers, total VFA concentration, and molar proportions of acetate and propionate were similar between cows fed BMCS and those fed CCS. Daily enteric CH4 emission (g/d) was unaffected by dietary treatments, but CH4 production expressed as a proportion of gross energy intake or on milk yield basis was lower for cows fed the BMCS-based diet than for cows fed the CCS-based diet. A decline in manure N excretion and a shift in N excretion from urine to feces were observed when BMCS replaced CCS in the diet, suggesting reduced potential of manure N volatilization. Results from this study show that improving fiber quality of corn silage in dairy cow diets through using brown midrib trait cultivar can reduce enteric CH4 emissions as well as potential emissions of NH3 and N2O from manure. However, CH4 emissions during manure storage may increase due to excretion of degradable OM when BMCS diet is fed, which merits further investigation.

The maize brown midrib4 (bm4) gene encodes a functional folylpolyglutamate synthase.

Mutations in the brown midrib4 (bm4) gene affect the accumulation and composition of lignin in maize. Fine-mapping analysis of bm4 narrowed the candidate region to an approximately 105 kb interval on chromosome 9 containing six genes. Only one of these six genes, GRMZM2G393334, showed decreased expression in mutants. At least four of 10 Mu-induced bm4 mutant alleles contain a Mu insertion in the GRMZM2G393334 gene. Based on these results, we concluded that GRMZM2G393334 is the bm4 gene. GRMZM2G393334 encodes a putative folylpolyglutamate synthase (FPGS), which functions in one-carbon (C1) metabolism to polyglutamylate substrates of folate-dependent enzymes. Yeast complementation experiments demonstrated that expression of the maize bm4 gene in FPGS-deficient met7 yeast is able to rescue the yeast mutant phenotype, thus demonstrating that bm4 encodes a functional FPGS. Consistent with earlier studies, bm4 mutants exhibit a modest decrease in lignin concentration and an overall increase in the S:G lignin ratio relative to wild-type. Orthologs of bm4 include at least one paralogous gene in maize and various homologs in other grasses and dicots. Discovery of the gene underlying the bm4 maize phenotype illustrates a role for FPGS in lignin biosynthesis.

Photosynthesis-related characteristics of the midrib and the interveinal lamina in leaves of the C3-CAM intermediate plant Mesembryanthemum crystallinum.

BACKGROUND AND AIMS: Leaf veins are usually encircled by specialized bundle sheath cells. In C4 plants, they play an important role in CO2 assimilation, and the photosynthetic activity is compartmentalized between the mesophyll and the bundle sheath. In C3 and CAM (Crassulacean acid metabolism) plants, the photosynthetic activity is generally attributed to the leaf mesophyll cells, and the vascular parenchymal cells are rarely considered for their role in photosynthesis. Recent studies demonstrate that enzymes required for C4 photosynthesis are also active in the veins of C3 plants, and their vascular system contains photosynthetically competent parenchyma cells. However, our understanding of photosynthesis in veins of C3 and CAM plants still remains insufficient. Here spatial analysis of photosynthesis-related properties were applied to the midrib and the interveinal lamina cells in leaves of Mesembryanthemum crystallinum, a C3-CAM intermediate plant. METHODS: The midrib anatomy as well as chloroplast structure and chlorophyll fluorescence, diurnal gas exchange profiles, the immunoblot patterns of PEPC (phosphoenolpyruvate carboxylase) and RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), H2O2 localization and antioxidant enzyme activities were compared in the midrib and in the interveinal mesophyll cells in leaves of C3 and CAM plants. KEY RESULTS: Leaf midribs were structurally competent to perform photosynthesis in C3 and CAM plants. The midrib chloroplasts resembled those in the bundle sheath cells of C4 plants and were characterized by limited photosynthetic activity. CONCLUSIONS: The metabolic roles of midrib chloroplasts differ in C3 and CAM plants. It is suggested that in leaves of C3 plants the midrib chloroplasts could be involved in the supply of CO2 for carboxylation, and in CAM plants they could provide malate to different metabolic processes and mediate H2O2 signalling.

The maize brown midrib2 (bm2) gene encodes a methylenetetrahydrofolate reductase that contributes to lignin accumulation.

The midribs of maize brown midrib (bm) mutants exhibit a reddish-brown color associated with reductions in lignin concentration and alterations in lignin composition. Here, we report the mapping, cloning, and functional and biochemical analyses of the bm2 gene. The bm2 gene was mapped to a small region of chromosome 1 that contains a putative methylenetetrahydrofolate reductase (MTHFR) gene, which is down-regulated in bm2 mutant plants. Analyses of multiple Mu-induced bm2-Mu mutant alleles confirmed that this constitutively expressed gene is bm2. Yeast complementation experiments and a previously published biochemical characterization show that the bm2 gene encodes a functional MTHFR. Quantitative RT-PCR analyses demonstrated that the bm2 mutants accumulate substantially reduced levels of bm2 transcript. Alteration of MTHFR function is expected to influence accumulation of the methyl donor S-adenosyl-L-methionine (SAM). Because SAM is consumed by two methyltransferases in the lignin pathway (Ye et al., ), the finding that bm2 encodes a functional MTHFR is consistent with its lignin phenotype. Consistent with this functional assignment of bm2, the expression patterns of genes in a variety of SAM-dependent or -related pathways, including lignin biosynthesis, are altered in the bm2 mutant. Biochemical assays confirmed that bm2 mutants accumulate reduced levels of lignin with altered composition compared to wild-type. Hence, this study demonstrates a role for MTHFR in lignin biosynthesis.

The effect of hybrid type and dietary proportions of corn silage on the lactation performance of high-producing dairy cows.

We evaluated the effects of corn silage hybrids [control vs. brown midrib (BMR)] and the proportion of corn silage in rations on the performance of high-producing dairy cows. The chemical composition of the corn silages was similar except for lignin, which was higher in the control hybrid [3.09%, dry matter (DM) basis] compared with the BMR hybrid (2.19%). The 30-h in vitro neutral detergent fiber (NDF) digestibility was also higher (62.8% of NDF) in the BMR hybrid than in the control hybrid (52.2%). Twenty-seven Holstein cows were fed 1 of 3 diets comprising 62% forage and 38% concentrate (DM basis) containing 35% (DM basis) corn silage from the control hybrid (NLO), 35% of the BMR hybrid (BLO), or 50% of the BMR (BHI). Cows were fed the diets in a replicated 3×3 Latin square design with 28-d periods. Intake of DM was similar among treatments but milk production was greater for cows fed BLO (50.1kg/d) and BHI (51.1kg/d) than for NLO (47.9kg/d). Milk fat percentage was lower for cows fed BHI (3.37%) than for those fed BLO (3.55%) and NLO (3.56%) but yield of milk fat was similar among treatments. Yield and percentage of milk protein was higher for cows in BHI compared with NLO. The concentration of milk urea N was lower in cows fed BHI (14.0mg/dL) than in those fed NLO (14.7mg/dL) and intermediate for BLO (14.5mg/dL). The yield of 3.5% fat-corrected milk was higher in cows fed BLO (50.2kg/d) than in NLO (48.2kg/d) and was intermediate for BHI (49.8kg/d). The total-tract digestibility of dietary DM, organic matter, starch, and crude protein was lower for cows in NLO compared with the other treatments. The total-tract digestibility of NDF was highest for BHI (54.4%), intermediate for BLO (50.9%), and lowest for NLO (43.2%). We conclude that BMR corn silage can be included in rations at moderate and high proportions of a total ration, resulting in high levels of milk production.

Acid detergent lignin, lodging resistance index, and expression of the caffeic acid O-methyltransferase gene in brown midrib-12 sudangrass.

Understanding the relationship between acid detergent lignin (ADL) and lodging resistance index (LRI) is essential for breeding new varieties of brown midrib (bmr) sudangrass (Sorghum sudanense (Piper) Stapf.). In this study, bmr-12 near isogenic lines and their wild-types obtained by back cross breeding were used to compare relevant forage yield and quality traits, and to analyze expression of the caffeic acid O-methyltransferase (COMT) gene using quantitative real time-PCR. The research showed that the mean ADL content of bmr-12 mutants (20.94 g kg(-1)) was significantly (P < 0.05) lower than measured in N-12 lines (43.45 g kg(-1)), whereas the LRI of bmr-12 mutants (0.29) was significantly (P < 0.05) higher than in N-12 lines (0.22). There was no significant correlation between the two indexes in bmr-12 materials (r = -0.44, P > 0.05). Sequence comparison of the COMT gene revealed two point mutations present in bmr-12 but not in the wild-type, the second mutation changed amino acid 129 of the protein from Gln (CAG) to a stop codon (UAG). The relative expression level of COMT gene was significantly reduced, which likely led to the decreased ADL content observed in the bmr-12 mutant.

Influence of corn silage hybrid type on lactation performance by Holstein dairy cows.

The primary objective of this study was to determine lactation performance by dairy cows fed nutridense (ND), dual-purpose (DP), or brown midrib (BM) corn silage hybrids at the same concentration in the diets. A secondary objective was to determine lactation performance by dairy cows fed NutriDense corn silage at a higher concentration in the diet. One hundred twenty-eight Holstein and Holstein × Jersey cows (105 ± 38 d in milk) were stratified by breed and parity and randomly assigned to 16 pens of 8 cows each. Pens were then randomly assigned to 1 of 4 treatments. Three treatment total mixed rations (TMR; DP40, BM40, and ND40) contained 40% of dry matter (DM) from the respective corn silage hybrid and 20% of DM from alfalfa silage. The fourth treatment TMR had ND corn silage as the sole forage at 65% of DM (ND65). A 2-wk covariate adjustment period preceded the treatment period, with all pens receiving a TMR with equal proportions of DP40, BM40, and ND40. Following the covariate period, cows were fed their assigned treatment diets for 11 wk. nutridense corn silage had greater starch and lower neutral detergent fiber (NDF) content than DP or BM, resulting in ND40 having greater energy content (73.2% of total digestible nutrients, TDN) than DP40 or BM40 (71.9 and 71.4% TDN, respectively). Cows fed BM40 had greater milk yield than DP40, whereas ND40 tended to have greater milk yield and had greater protein and lactose yields compared with DP40. No differences in intake, component-corrected milk yields, or feed efficiency were detected between DP40, BM40, and ND40. Milk yield differences may be due to increased starch intake for ND40 and increased digestible NDF intake for BM40 compared with DP40. Intake and milk yield and composition were similar for ND40 compared with BM40, possibly due to counteracting effects of higher starch intake for ND40 and higher digestible NDF intake for BM40. Feeding ND65 reduced intake, and thus milk and component yields, compared with ND40 due to either increased ruminal starch digestibility or increased rumen fill for ND65. Nutridense corn silage was a viable alternative to both DP and BM at 40% of diet DM; however, lactation performance was reduced when nutridense corn silage was fed at 65% of DM.