High-density genetic map construction and QTL mapping of a zigzag-shaped stem trait in tea plant (Camellia sinensis).

The highly unique zigzag-shaped stem phenotype in tea plants boasts significant ornamental value and is exceptionally rare. To investigate the genetic mechanism behind this trait, we developed BC1 artificial hybrid populations. Our genetic analysis revealed the zigzag-shaped trait as a qualitative trait. Utilizing whole-genome resequencing, we constructed a high-density genetic map from the BC1 population, incorporating 5,250 SNP markers across 15 linkage groups, covering 3,328.51 cM with an average marker interval distance of 0.68 cM. A quantitative trait locus (QTL) for the zigzag-shaped trait was identified on chromosome 4, within a 61.2 to 97.2 Mb range, accounting for a phenotypic variation explained (PVE) value of 13.62%. Within this QTL, six candidate genes were pinpointed. To better understand their roles, we analyzed gene expression in various tissues and individuals with erect and zigzag-shaped stems. The results implicated CsXTH (CSS0035625) and CsCIPK14 (CSS0044366) as potential key contributors to the zigzag-shaped stem formation. These discoveries lay a robust foundation for future functional genetic mapping and tea plant genetic enhancement.

Starch and sucrose metabolism plays an important role in the stem development in Medicago sativa.

The forage quality of alfalfa (Medicago sativa ) stems is greater than the leaves. Sucrose hydrolysis provides energy for stem development, with starch being enzymatically converted into sucrose to maintain energy homeostasis. To understand the physiological and molecular networks controlling stem development, morphological characteristics and transcriptome profiles in the stems of two alfalfa cultivars (Zhungeer and WL168) were investigated. Based on transcriptome data, we analysed starch and sugar contents, and enzyme activity related to starch-sugar interconversion. Zhungeer stems were shorter and sturdier than WL168, resulting in significantly higher mechanical strength. Transcriptome analysis showed that starch and sucrose metabolism were significant enriched in the differentially expressed genes of stems development in both cultivars. Genes encoding INV , bglX , HK , TPS and glgC downregulated with the development of stems, while the gene encoding was AMY upregulated. Weighted gene co-expression network analysis revealed that the gene encoding glgC was pivotal in determining the variations in starch and sucrose contents between the two cultivars. Soluble carbohydrate, sucrose, and starch content of WL168 were higher than Zhungeer. Enzyme activities related to sucrose synthesis and hydrolysis (INV, bglX, HK, TPS) showed a downward trend. The change trend of enzyme activity was consistent with gene expression. WL168 stems had higher carbohydrate content than Zhungeer, which accounted for more rapid growth and taller plants. WL168 formed hollow stems were formed during rapid growth, which may be related to the redistribution of carbohydrates in the pith tissue. These results indicated that starch and sucrose metabolism play important roles in the stem development in alfalfa.

Bulked Segregant RNA-Seq Reveals Different Gene Expression Patterns and Mutant Genes Associated with the Zigzag Pattern of Tea Plants (Camellia sinensis).

The unique zigzag-patterned tea plant is a rare germplasm resource. However, the molecular mechanism behind the formation of zigzag stems remains unclear. To address this, a BC1 genetic population of tea plants with zigzag stems was studied using histological observation and bulked segregant RNA-seq. The analysis revealed 1494 differentially expressed genes (DEGs) between the upright and zigzag stem groups. These DEGs may regulate the transduction and biosynthesis of plant hormones, and the effects on the phenylpropane biosynthesis pathways may cause the accumulation of lignin. Tissue sections further supported this finding, showing differences in cell wall thickness between upright and curved stems, potentially due to lignin accumulation. Additionally, 262 single-nucleotide polymorphisms (SNPs) across 38 genes were identified as key SNPs, and 5 genes related to zigzag stems were identified through homologous gene function annotation. Mutations in these genes may impact auxin distribution and content, resulting in the asymmetric development of vascular bundles in curved stems. In summary, we identified the key genes associated with the tortuous phenotype by using BSR-seq on a BC1 population to minimize genetic background noise.

Identification and characterization of PAL genes involved in the regulation of stem development in Saccharum spontaneum L.

BACKGROUND: Saccharum spontaneum L. is a closely related species of sugarcane and has become an important genetic component of modern sugarcane cultivars. Stem development is one of the important factors for affecting the yield, while the molecular mechanism of stem development remains poorly understanding in S. spontaneum. Phenylalanine ammonia-lyase (PAL) is a vital component of both primary and secondary metabolism, contributing significantly to plant growth, development and stress defense. However, the current knowledge about PAL genes in S. spontaneum is still limited. Thus, identification and characterization of the PAL genes by transcriptome analysis will provide a theoretical basis for further investigation of the function of PAL gene in sugarcane. RESULTS: In this study, 42 of PAL genes were identified, including 26 SsPAL genes from S. spontaneum, 8 ShPAL genes from sugarcane cultivar R570, and 8 SbPAL genes from sorghum. Phylogenetic analysis showed that SsPAL genes were divided into three groups, potentially influenced by long-term natural selection. Notably, 20 SsPAL genes were existed on chromosomes 4 and 5, indicating that they are highly conserved in S. spontaneum. This conservation is likely a result of the prevalence of whole-genome replications within this gene family. The upstream sequence of PAL genes were found to contain conserved cis-acting elements such as G-box and SP1, GT1-motif and CAT-box, which collectively regulate the growth and development of S. spontaneum. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that SsPAL genes of stem had a significantly upregulated than that of leaves, suggesting that they may promote the stem growth and development, particularly in the + 6 stem (The sixth cane stalk from the top to down) during the growth stage. CONCLUSIONS: The results of this study revealed the molecular characteristics of SsPAL genes and indicated that they may play a vital role in stem growth and development of S. spontaneum. Altogether, our findings will promote the understanding of the molecular mechanism of S. spontaneum stem development, and also contribute to the sugarcane genetic improving.

Overexpression of tocopherol biosynthesis genes in guayule (Parthenium argentatum) reduces rubber, resin and argentatins content in stem and leaf tissues.

Natural rubber produced in stems of the guayule plant (Parthenium argentatum) is susceptible to post-harvest degradation from microbial or thermo-oxidative processes, especially once stems are chipped. As a result, the time from harvest to extraction must be minimized to recover high quality rubber, especially in warm summer months. Tocopherols are natural antioxidants produced in plants through the shikimate and methyl-erythtiol-4-phosphate (MEP) pathways. We hypothesized that increased in vivo guayule tocopherol content might protect rubber from post-harvest degradation, and/or allow reduced use of chemical antioxidants during the extraction process. With the objective of enhancing tocopherol content in guayule, we overexpressed four Arabidopsis thaliana tocopherol pathway genes in AZ-2 guayule via Agrobacterium-mediated transformation. Tocopherol content was increased in leaf and stem tissues of most transgenic lines, and some improvement in thermo-oxidative stability was observed. Overexpression of the four tocopherol biosynthesis enzymes, however, altered other isoprenoid pathways resulting in reduced rubber, resin and argentatins content in guayule stems. The latter molecules are mainly synthesized from precursors derived from the mevalonate (MVA) pathway. Our results suggest the existence of crosstalk between the MEP and MVA pathways in guayule and the possibility that carbon metabolism through the MEP pathway impacts rubber biosynthesis.

Salicylic acid and RNA interference mediate antiviral immunity of plant stem cells.

Stem cells are essential for the development and organ regeneration of multicellular organisms, so their infection by pathogenic viruses must be prevented. Accordingly, mammalian stem cells are highly resistant to viral infection due to dedicated antiviral pathways including RNA interference (RNAi). In plants, a small group of stem cells harbored within the shoot apical meristem generate all postembryonic above-ground tissues, including the germline cells. Many viruses do not proliferate in these cells, yet the molecular bases of this exclusion remain only partially understood. Here, we show that a plant-encoded RNA-dependent RNA polymerase, after activation by the plant hormone salicylic acid, amplifies antiviral RNAi in infected tissues. This provides stem cells with RNA-based virus sequence information, which prevents virus proliferation. Furthermore, we find RNAi to be necessary for stem cell exclusion of several unrelated RNA viruses, despite their ability to efficiently suppress RNAi in the rest of the plant. This work elucidates a molecular pathway of great biological and economic relevance and lays the foundations for our future understanding of the unique systems underlying stem cell immunity.

Transcription factor NTL9 negatively regulates Arabidopsis vascular cambium development during stem secondary growth.

In plant stems, secondary vascular development is established through the differentiation of cylindrical vascular cambium, producing secondary xylem (wood) and phloem (bast), which have economic importance. However, there is a dearth of knowledge on the genetic mechanism underlying this process. NAC with Transmembrane Motif 1-like transcription factor 9 (NTL9) plays a central role in abiotic and immune signaling responses. Here, we investigated the role of NTL9 in vascular cambium development in Arabidopsis (Arabidopsis thaliana) inflorescence stems by identifying and characterizing an Arabidopsis phloem circular-timing (pct) mutant. The pct mutant exhibited enhanced vascular cambium formation following secondary phloem production. In the pct mutant, although normal organization in vascular bundles was maintained, vascular cambium differentiation occurred at an early stage of stem development, which was associated with increased expression of cambium-/phloem-related genes and enhanced cambium activity. The pct mutant stem phenotype was caused by a recessive frameshift mutation that disrupts the transmembrane (TM) domain of NTL9. Our results indicate that NTL9 functions as a negative regulator of cambial activity and has a suppressive role in developmental transition to the secondary growth phase in stem vasculature, which is necessary for its precise TM domain-mediated regulation.

Dynamic evolution of small signalling peptide compensation in plant stem cell control.

Gene duplications are a hallmark of plant genome evolution and a foundation for genetic interactions that shape phenotypic diversity1-5. Compensation is a major form of paralogue interaction6-8 but how compensation relationships change as allelic variation accumulates is unknown. Here we leveraged genomics and genome editing across the Solanaceae family to capture the evolution of compensating paralogues. Mutations in the stem cell regulator CLV3 cause floral organs to overproliferate in many plants9-11. In tomato, this phenotype is partially suppressed by transcriptional upregulation of a closely related paralogue12. Tobacco lost this paralogue, resulting in no compensation and extreme clv3 phenotypes. Strikingly, the paralogues of petunia and groundcherry nearly completely suppress clv3, indicating a potent ancestral state of compensation. Cross-species transgenic complementation analyses show that this potent compensation partially degenerated in tomato due to a single amino acid change in the paralogue and cis-regulatory variation that limits its transcriptional upregulation. Our findings show how genetic interactions are remodelled following duplications and suggest that dynamic paralogue evolution is widespread over short time scales and impacts phenotypic variation from natural and engineered mutations.

Adult plant stem rust resistance in durum wheat Glossy Huguenot: mapping, marker development and validation.

KEY MESSAGE: Adult plant stem rust resistance locus, QSrGH.cs-2AL, was identified in durum wheat Glossy Huguenot and mendelised as Sr63. Markers closely linked with Sr63 were developed. An F3 population from a Glossy Huguenot (GH)/Bansi cross used in a previous Australian study was advanced to F6 for molecular mapping of adult plant stem rust resistance. Maturity differences among F6 lines confounded assessments of stem rust response. GH was crossed with a stem rust susceptible F6 recombinant inbred line (RIL), GHB14 (M14), with similar maturity and an F6:7 population was developed through single seed descent method. F7 and F8 RILs were tested along with the parents at different locations. The F6 individual plants and both parents were genotyped using the 90 K single nucleotide polymorphism (SNP) wheat array. Stem rust resistance QTL on the long arms of chromosomes 1B (QSrGH.cs-1BL) and 2A (QSrGH.cs-2AL) were detected. QSrGH.cs-1BL and QSrGH.cs-2AL were both contributed by GH and explained 22% and 18% adult plant stem rust response variation, respectively, among GH/M14 RIL population. RILs carrying combinations of these QTL reduced more than 14% stem rust severity compared to those that possessed QSrGH.cs-1BL and QSrGH.cs-2AL individually. QSrGH.cs1BL was demonstrated to be the same as Sr58/Lr46/Yr29/Pm39 through marker genotyping. Lines lacking QSrGH.cs-1BL were used to Mendelise QSrGH.cs-2AL. Based on genomic locations of previously catalogued stem rust resistance genes and the QSrGH.cs-2AL map, it appeared to represent a new APR locus and was permanently named Sr63. SNP markers associated with Sr63 were converted to kompetetive allele-specific PCR (KASP) assays and were validated on a set of durum cultivars.

Study on population structure of kiwifruit and GWAS for hairiness character.

BACKGROUND: A total of 74,936 SNPs were employed to carry out population structure and genome-wide association studies and post-GWAS for hairiness character of the fifty-six samples including thirty-six Actinidia chinensis var. deliciosa, eighteen A. chinensis var. chinensis, and two A. polygama in the light of morphological observations. RESULTS: The percentage of heterozygous sites of A. chinensis var. deliciosa is higher than that of A. chinensis var. chinensis, which could be one of the reasons for A. chinensis var. deliciosa high disease resistance. Fifty-six samples were divided into two subgroups, in which the genetic distance, ranged from 0.17 to 0.99, according to their genetic divergence. Analysis of molecular variance shows that the frequency of genetic variations within the population is 83.53% and 16.47% between populations. Fst between the two populations is 0.14, and Nm is 1.60. Set at α ≤ 0.05, a total of 327 SNPs and 260 haplotypes were related to the hairiness character. A total of 246 proteins were annotated using GO and KEGG analyses, which indicated the membrane-related genes and stress-resistant metabolic pathways are related to the hairiness character of leaves, stems, and peels of kiwifruit. Protein interaction analysis showed that DNA-directed RNA polymerase was an important node protein that interacted with many proteins. CONCLUSIONS: The genetic basic in the fifty-six genotypes was rich. The results of clustering and morphological observations are not completely consistent, indicating the hairiness character play an important role in the classification of kiwifruit, in which two A. polygama were clustered together with those of A. chinensis var. chinensis. Phylogeny and haplotype analysis showed that the evolution of A. chinensis var. chinensis is later than that of A. chinensis var. deliciosa in A. chinesis. The loss of hairiness character on leaves, stems and peels of A. chinensis var. chinensis compare with A. chinensis var. deliciosa, which is also the result of its poor resistance.

Molecular evolution and functional characterization of chitinase gene family in Populus trichocarpa.

Chitinases, the chitin-degrading enzymes, have been shown to play important role in defense against the chitin-containing fungal pathogens. In this study, we identified 48 chitinase-coding genes from the woody model plant Populus trichocarpa. Based on phylogenetic analysis, the Populus chitinases were classified into seven groups. Different gene structures and protein domain architectures were found among the seven Populus chitinase groups. Selection pressure analysis indicated that all the seven groups are under purifying selection. Phylogenetic analysis combined with chromosome location analysis showed that Populus chitinase gene family mainly expanded through tandem duplication. The Populus chitinase gene family underwent marked expression divergence and is inducibly expressed in response to treatments, such as chitosan, chitin, salicylic acid and methyl jasmonate. Protein enzymatic activity analysis showed that Populus chitinases had activity towards both chitin and chitosan. By integrating sequence characteristic, phylogenetic, selection pressure, gene expression and protein activity analysis, this study shed light on the evolution and function of chitinase family in poplar.

An Efficient Marker Gene Excision Strategy Based on CRISPR/Cas9-Mediated Homology-Directed Repair in Rice.

In order to separate transformed cells from non-transformed cells, antibiotic selectable marker genes are usually utilized in genetic transformation. After obtaining transgenic plants, it is often necessary to remove the marker gene from the plant genome in order to avoid regulatory issues. However, many marker-free systems are time-consuming and labor-intensive. Homology-directed repair (HDR) is a process of homologous recombination using homologous arms for efficient and precise repair of DNA double-strand breaks (DSBs). The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 (Cas9) system is a powerful genome editing tool that can efficiently cause DSBs. Here, we isolated a rice promoter (Pssi) of a gene that highly expressed in stem, shoot tip and inflorescence, and established a high-efficiency sequence-excision strategy by using this Pssi to drive CRISPR/Cas9-mediated HDR for marker free (PssiCHMF). In our study, PssiCHMF-induced marker gene deletion was detected in 73.3% of T0 plants and 83.2% of T1 plants. A high proportion (55.6%) of homozygous marker-excised plants were obtained in T1 progeny. The recombinant GUS reporter-aided analysis and its sequencing of the recombinant products showed precise deletion and repair mediated by the PssiCHMF method. In conclusion, our CRISPR/Cas9-mediated HDR auto-excision method provides a time-saving and efficient strategy for removing the marker genes from transgenic plants.

Transcriptome analysis reveals the mechanism of internode development affecting maize stalk strength.

BACKGROUND: The stalk rind is one of the important factors affecting maize stalk strength that is closely related to stalk lodging. However, the mechanism of rind development in maize is still largely unknown. RESULTS: In this study, we analyzed the mechanical, anatomical, and biochemical properties of the third basal internode in one maize non-stiff-stalk (NSS) line and two stiff-stalk (SS) lines. Compared with the NSS line, the two SS lines had a significantly higher rind penetrometer resistance, thicker rind, and higher dry matter, hemicellulose, cellulose, and lignin weights per unit length. RNA-seq analysis was used to compare transcriptomes of the third basal internode of the two SS lines and the NSS line at the ninth leaf and tasseling stages. Gene Ontology (GO) enrichment analysis revealed that genes involved in hydrolase activity (hydrolyzing O-glycosyl compounds) and cytoskeleton organization were significantly up-regulated in the two SS lines at the ninth leaf stage and that microtubule process-related genes were significantly up-regulated in the two SS lines at the tasseling stage. Moreover, the two SS lines had enhanced expression of cell wall metabolism-related genes at the tasseling stage. CONCLUSIONS: The synthesis of cell wall polysaccharides and the cytoskeleton might play important roles in internode development. Our results can be applied for screening lodging-resistant inbred lines and breeding lodging-resistant cultivars in maize.

Proteomics of isolated sieve tubes from Nicotiana tabacum: sieve element-specific proteins reveal differentiation of the endomembrane system.

Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins ( SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.

Natural variation of ZmCGT1 is responsible for isoorientin accumulation in maize silk.

Maize (Zea mays L.) silk contains high levels of flavonoids and is widely used to promote human health. Isoorientin, a natural C-glycoside flavone abundant in maize silk, has attracted considerable attention due to its potential value. Although different classes of flavonoid have been well characterized in plants, the genes involved in the biosynthesis of isoorientin in maize are largely unknown. Here, we used targeted metabolic profiling of isoorientin on the silks in an association panel consisting of 294 maize inbred lines. We identified the gene ZmCGT1 by genome-wide association analysis. The ZmCGT1 protein was characterized as a 2-hydroxyflavanone C-glycosyltransferase that can C-glycosylate 2-hydroxyflavanone to form flavone-C-glycoside after dehydration. Moreover, ZmCGT1 overexpression increased isoorientin levels and RNA interference-mediated ZmCGT1 knockdown decreased accumulation of isoorientin in maize silk. Further, two nucleotide polymorphisms, A502C and A1022G, which led to amino acid changes I168L and E341G, respectively, were identified to be functional polymorphisms responsible for the natural variation in isoorientin levels. In summary, we identified the gene ZmCGT1, which plays an important role in isoorientin biosynthesis, providing insights into the genetic basis of the natural variation in isoorientin levels in maize silk. The identified favorable CG allele of ZmCGT1 may be further used for genetic improvement of nutritional quality in maize.

Knockout of the lignin pathway gene BnF5H decreases the S/G lignin compositional ratio and improves Sclerotinia sclerotiorum resistance in Brassica napus.

Ferulate-5-hydroxylase is a key enzyme involved in the conversion of the guaiacyl monolignol to the syringyl monolignol in angiosperms. The monolignol ratio has been proposed to affect biomass recalcitrance and the resistance to plant disease. Stem rot caused by the fungus Sclerotinia sclerotiorum in Brassica napus causes severe losses in its production. To date, there is no information about the effect of the lignin monomer ratio on the resistance to S. sclerotiorum in B. napus. Four dominantly expressed ferulate-5-hydroxylase genes were concertedly knocked out by CRISPR/Cas9 in B. napus, and three mutant lines were generated. The S/G lignin compositional ratio was decreased compared to that of the wild type based on the results of Mӓule staining and 2D-NMR profiling in KO-7. The resistance to S. sclerotiorum in stems and leaves increased for the three f5h mutant lines compared with WT. Furthermore, we found that the stem strength of f5h mutant lines was significantly increased. Overall, we demonstrate for the first time that decreasing the S/G ratio by knocking out of the F5H gene improves S. sclerotiorum resistance in B. napus and increases stem strength.

Quantitative Inheritance of Sclerotinia Stem Rot Resistance in Brassica napus and Relationship to Cotyledon and Leaf Resistances.

Sclerotinia sclerotiorum is a necrotrophic fungus causing devastating stem rot and associated yield losses of canola/rapeseed (Brassica napus) worldwide, including in Australia. Developing host resistance against Sclerotinia stem rot is critical if this disease in canola/rapeseed is to be successfully managed, as cultural or chemical control options provide only partial or sporadic control. Three B. napus breeding populations, C2, C5 and C6, including the parents, F1, F2, BC1P1, and BC2P2, were used in a field study with an objective of exploring the inheritance pattern of disease resistance (based on stem lesion length [SLL]) and the genetic relationships of disease with stem diameter (SD) or days to first flowering (DTF), and to compare these new adult plant stem resistances against S. sclerotiorum with those of seedling (cotyledon and leaf) resistances in earlier studies. Heritability (broad sense) of SLL was 0.57 and 0.73 for population C2 at 3 and 5 weeks postinoculation and 0.21 for population C5 at 5 weeks postinoculation. Additive genetic variance was evident within all 3 populations for DTF but not for SD. Narrow-sense heritability for DTF was 0.48 (C2), 0.42 (C5), and 0.32 (C6). SD, DTF, and SLL were all inherited independently, with no significant genetic covariance between traits in bivariate analysis. Genetic variance for SLL in populations C2 and C5 was entirely nonadditive, and there was significant nonadditive genetic covariance of SLL at 3 and 5 weeks postinoculation. Generation means analysis in population C2 supported the conclusion that complex epistatic interactions controlled SLL. Several C2 and C5 progeny showed high adult plant stem resistance, which may be critical in developing enhanced stem resistance in canola/rapeseed. Although population C6 showed no genetic variation for SLL resistance in this study, it showed significant nonadditive genetic variance at the cotyledon and leaf stages in earlier studies. We conclude that host resistance varies across different plant growth stages, and breeding must be targeted for resistance at each growth stage. In populations C2, C5, and C6, resistance to S. sclerotiorum in stem, leaf, and cotyledon was always controlled by nonadditive effects such as complex epistasis or dominance. Overall, our findings in relation to the quantitative inheritance of Sclerotinia stem rot resistance, together with the new high-level resistances identified, will enable breeders to select/develop genotypes with enhanced resistances to S. sclerotiorum.

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

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

Planting Season Impacts Sugarcane Stem Development, Secondary Metabolite Levels, and Natural Antisense Transcription.

To reduce the potentially irreversible environmental impacts caused by fossil fuels, the use of renewable energy sources must be increased on a global scale. One promising source of biomass and bioenergy is sugarcane. The study of this crop's development in different planting seasons can aid in successfully cultivating it in global climate change scenarios. The sugarcane variety SP80-3280 was field grown under two planting seasons with different climatic conditions. A systems biology approach was taken to study the changes on physiological, morphological, agrotechnological, transcriptomics, and metabolomics levels in the leaf +1, and immature, intermediate and mature internodes. Most of the variation found within the transcriptomics and metabolomics profiles is attributed to the differences among the distinct tissues. However, the integration of both transcriptomics and metabolomics data highlighted three main metabolic categories as the principal sources of variation across tissues: amino acid metabolism, biosynthesis of secondary metabolites, and xenobiotics biodegradation and metabolism. Differences in ripening and metabolite levels mainly in leaves and mature internodes may reflect the impact of contrasting environmental conditions on sugarcane development. In general, the same metabolites are found in mature internodes from both "one-year" and "one-and-a-half-year sugarcane", however, some metabolites (i.e., phenylpropanoids with economic value) and natural antisense transcript expression are only detected in the leaves of "one-year" sugarcane.