An ARF gene mutation creates flint kernel architecture in dent maize.

Dent and flint kernel architectures are important characteristics that affect the physical properties of maize kernels and their grain end uses. The genes controlling these traits are unknown, so it is difficult to combine the advantageous kernel traits of both. We found mutation of ARFTF17 in a dent genetic background reduces IAA content in the seed pericarp, creating a flint-like kernel phenotype. ARFTF17 is highly expressed in the pericarp and encodes a protein that interacts with and inhibits MYB40, a transcription factor with the dual functions of repressing PIN1 expression and transactivating genes for flavonoid biosynthesis. Enhanced flavonoid biosynthesis could reduce the metabolic flux responsible for auxin biosynthesis. The decreased IAA content of the dent pericarp appears to reduce cell division and expansion, creating a shorter, denser kernel. Introgression of the ARFTF17 mutation into dent inbreds and hybrids improved their kernel texture, integrity, and desiccation, without affecting yield.

Spatial transcriptomics uncover sucrose post-phloem transport during maize kernel development.

Maize kernels are complex biological systems composed of three genetic sources, namely maternal tissues, progeny embryos, and progeny endosperms. The lack of gene expression profiles with spatial information has limited the understanding of the specific functions of each cell population, and hindered the exploration of superior genes in kernels. In our study, we conduct microscopic sectioning and spatial transcriptomics analysis during the grain filling stage of maize kernels. This enables us to visualize the expression patterns of all genes through electronical RNA in situ hybridization, and identify 11 cell populations and 332 molecular marker genes. Furthermore, we systematically elucidate the spatial storage mechanisms of the three major substances in maize kernels: starch, protein, and oil. These findings provide valuable insights into the functional genes that control agronomic traits in maize kernels.

THP9 enhances seed protein content and nitrogen-use efficiency in maize.

Teosinte, the wild ancestor of maize (Zea mays subsp. mays), has three times the seed protein content of most modern inbreds and hybrids, but the mechanisms that are responsible for this trait are unknown1,2. Here we use trio binning to create a contiguous haplotype DNA sequence of a teosinte (Zea mays subsp. parviglumis) and, through map-based cloning, identify a major high-protein quantitative trait locus, TEOSINTE HIGH PROTEIN 9 (THP9), on chromosome 9. THP9 encodes an asparagine synthetase 4 enzyme that is highly expressed in teosinte, but not in the B73 inbred, in which a deletion in the tenth intron of THP9-B73 causes incorrect splicing of THP9-B73 transcripts. Transgenic expression of THP9-teosinte in B73 significantly increased the seed protein content. Introgression of THP9-teosinte into modern maize inbreds and hybrids greatly enhanced the accumulation of free amino acids, especially asparagine, throughout the plant, and increased seed protein content without affecting yield. THP9-teosinte seems to increase nitrogen-use efficiency, which is important for promoting a high yield under low-nitrogen conditions.

AtSEC22 Regulates Cell Morphogenesis via Affecting Cytoskeleton Organization and Stabilities.

The plant cytoskeleton forms a stereoscopic network that regulates cell morphogenesis. The cytoskeleton also provides tracks for trafficking of vesicles to the target membrane. Fusion of vesicles with the target membrane is promoted by SNARE proteins, etc. The vesicle-SNARE, Sec22, regulates membrane trafficking between the ER and Golgi in yeast and mammals. Arabidopsis AtSEC22 might also regulate early secretion and is essential for gametophyte development. However, the role of AtSEC22 in plant development is unclear. To clarify the role of AtSEC22 in the regulation of plant development, we isolated an AtSEC22 knock-down mutant, atsec22-4, and found that cell morphogenesis and development were seriously disturbed. atsec22-4 exhibited shorter primary roots (PRs), dwarf plants, and partial abortion. More interestingly, the atsec22-4 mutant had less trichomes with altered morphology, irregular stomata, and pavement cells, suggesting that cell morphogenesis was perturbed. Further analyses revealed that in atsec22-4, vesicle trafficking was blocked, resulting in the trapping of proteins in the ER and collapse of structures of the ER and Golgi apparatus. Furthermore, AtSEC22 defects resulted in impaired organization and stability of the cytoskeleton in atsec22-4. Our findings revealed essential roles of AtSEC22 in membrane trafficking and cytoskeleton dynamics during plant development.

PtBRI1.2 promotes shoot growth and wood formation through a brassinosteroid-mediated PtBZR1-PtWNDs module in poplar.

Brassinosteroid-insensitive-1 (BRI1) plays important roles in various signalling pathways controlling plant growth and development. However, the regulatory mechanism of BRI1 in brassinosteroid (BR)-mediated signalling for shoot growth and wood formation in woody plants is largely unknown. In this study, PtBRI1.2, a brassinosteroid-insensitive-1 gene, was overexpressed in poplar. Shoot growth and wood formation of transgenic plants were examined and the regulatory genes involved were verified. PtBRI1.2 was localized to the plasma membrane, with a predominant expression in leaves. Ectopic expression of PtBRI1.2 in Arabidopsis bri1-201 and bri1-5 mutants rescued their retarded-growth phenotype. Overexpression of PtBRI1.2 in poplar promoted shoot growth and wood formation in transgenic plants. Further studies revealed that overexpression of PtBRI1.2 promoted the accumulation of PtBZR1 (BRASSINAZOLE RESISTANT1) in the nucleus, which subsequently activated PtWNDs (WOOD-ASSOCIATED NAC DOMAIN transcription factors) to up-regulate expression of secondary cell wall biosynthesis genes involved in wood formation. Our results suggest that PtBRI1.2 plays a crucial role in regulating shoot growth and wood formation by activating BR signalling.

The B3 domain-containing transcription factor ZmABI19 coordinates expression of key factors required for maize seed development and grain filling.

Grain filling in maize (Zea mays) is regulated by a group of spatiotemporally synchronized transcription factors (TFs), but the factors that coordinate their expression remain unknown. We used the promoter of the grain filling-specific TF gene Opaque2 (O2) to screen upstream regulatory factors and identified a B3 domain TF, ZmABI19, that directly binds to the O2 promoter for transactivation. zmabi19 mutants displayed developmental defects in the endosperm and embryo, and mature kernels were opaque and reduced in size. The accumulation of zeins, starch and lipids dramatically decreased in zmabi19 mutants. RNA sequencing revealed an alteration of the nutrient reservoir activity and starch and sucrose metabolism in zmabi19 endosperms, and plant phytohormone signal transduction and lipid metabolism in zmabi19 embryos. Chromatin immunoprecipitation followed by sequencing coupled with differential expression analysis identified 106 high-confidence direct ZmABI19 targets. ZmABI19 directly regulates multiple key grain filling TFs including O2, Prolamine-box binding factor 1, ZmbZIP22, NAC130, and Opaque11 in the endosperm and Viviparous1 in the embryo. A number of phytohormone-related genes were also bound and regulated by ZmABI19. Our results demonstrate that ZmABI19 functions as a grain filling initiation regulator. ZmABI19 roles in coupling early endosperm and embryo development are also discussed.

Carotenoids modulate kernel texture in maize by influencing amyloplast envelope integrity.

The mechanism that creates vitreous endosperm in the mature maize kernel is poorly understood. We identified Vitreous endosperm 1 (Ven1) as a major QTL influencing this process. Ven1 encodes ß-carotene hydroxylase 3, an enzyme that modulates carotenoid composition in the amyloplast envelope. The A619 inbred contains a nonfunctional Ven1 allele, leading to a decrease in polar and an increase in non-polar carotenoids in the amyloplast. Coincidently, the stability of amyloplast membranes is increased during kernel desiccation. The lipid composition in endosperm cells in A619 is altered, giving rise to a persistent amyloplast envelope. These changes impede the gathering of protein bodies and prevent them from interacting with starch grains, creating air spaces that cause an opaque kernel phenotype. Genetic modifiers were identified that alter the effect of Ven1A619, while maintaining a high ß-carotene level. These studies provide insight for breeding vitreous kernel varieties and high vitamin A content in maize.

PtiCYP85A3, a BR C-6 Oxidase Gene, Plays a Critical Role in Brassinosteroid-Mediated Tension Wood Formation in Poplar.

In angiosperm trees, the gelatinous layer (G-layer) takes a great part of the fiber cell wall in the tension wood (TW). However, the mechanism underlying G-layer formation in poplar is largely unknown. In this work, we demonstrate that G-layer formation in poplar TW cells is regulated by brassinosteroid (BR) and its signaling. PtiCYP85A3, a key BR biosynthesis gene, was predominantly expressed in the xylem of TW, accompanied with a relatively higher castasterone (CS) accumulation, than in the xylem of opposite wood (OW). A wider expression zone of BZR1, a key transcriptional factor in BR singling pathway, was also observed in G-fiber cells on TW side than in wood fiber cells on the OW side, as indicated by immunohistochemistry assays. Transgenic poplar plants overexpressing PtiCYP85A3 produced thicker G-layer with higher cellulose proportion, and accumulated more BZR1 protein in the xylem of TW than did the wild type (WT) plants. Expression of most TW-associated CesAs, which were induced by 2, 4-epibrassinolide, an active BR, and inhibited by brassinazole, a BR biosynthesis inhibitor, were also up-regulated in the xylem of TW in transgenic plants compared to that in WT plants. Further studies with dual-luciferase assays demonstrated that the promoters of PtiCesAs were activated by PtiMYB128, a TW specific transcription factor, which was then regulated by BZR1. All these results indicate that BR plays a crucial role in the G-layer formation of TW fiber cells by regulating the expression of BZR1, PtiMYB128, and PtiCesAs in poplar.

MYB transcription factor PdMYB118 directly interacts with bHLH transcription factor PdTT8 to regulate wound-induced anthocyanin biosynthesis in poplar.

BACKGROUND: R2R3-MYB transcription factors (TFs) play important roles in plant growth and development, and response to biotic and abiotic stresses. However, their regulatory mechanisms in wound-induced anthocyanin biosynthesis in woody plants are largely unknown. RESULTS: In this work, we report that expression of anthocyanin biosynthesis genes (ABGs) were activated by PdMYB118, a MYB TF encoding gene from Populus deltoids, and the activation of PdMYB118 was significantly enhanced by PdTT8, a bHLH protein, through its direct interaction with PdMYB118. PdMYB118 and some ABGs were evidently induced by wound induction and methyl jasmonate (MeJA) treatment. Overexpression of PdMYB118 promoted anthocyanin accumulation in transgenic poplar upon wound induction. Furthermore, a poplar JASMONATE ZIM-domain (JAZ) protein, PtrJAZ1, repressed the transcriptional function of PdMYB118/PdTT8 complex by binding to PdTT8, and wound stimulated the biosynthesis of jasmonic acid (JA) and the degradation of PtrJAZ1. CONCLUSIONS: Based on these observations, we proposed that PtrJAZ1 degradation triggered the expression of ABGs, leading to increased biosynthesis of anthocyanins in the wounded leaves of transgenic poplar. Therefore, our findings not only illustrate the crucial role of PdMYB118 in wound-induced anthocyanin biosynthesis in poplar, but also provide a molecular basis for the genetic engineering of colorful tree species.

High frequency DNA rearrangement at qγ27 creates a novel allele for Quality Protein Maize breeding.

Copy number variation (CNV) is a major source of genetic variation and often contributes to phenotypic variation in maize. The duplication at the 27-kDa γ-zein locus (qγ27) is essential to convert soft endosperm into hard endosperm in quality protein maize (QPM). This duplication is unstable and generally produces CNV at this locus. We conducted genetic experiments designed to directly measure DNA rearrangement frequencies occurring in males and females of different genetic backgrounds. The average frequency with which the duplication rearranges to single copies is 1.27 × 10-3 and varies among different lines. A triplication of γ27 gene was screened and showed a better potential than the duplication for the future QPM breeding. Our results highlight a novel approach to directly determine the frequency of DNA rearrangements, in this case resulting in CNV at the qγ27 locus. Furthermore, this provides a highly effective way to test suitable parents in QPM breeding.

PdMYB118, isolated from a red leaf mutant of Populus deltoids, is a new transcription factor regulating anthocyanin biosynthesis in poplar.

KEY MESSAGE: A new anthocyanin biosynthesis transcription factor PdMYB118, which could be used for the genetic engineering of colorful tree species, was indentified from a red leaf mutant of Populus deltoids. In higher plants, the biosynthesis of anthocyanins is regulated by several classes of transcription factors (TFs), including R2R3-MYB, bHLH and WD-repeat proteins. In this work, we isolated an MYB gene regulating anthocyanin biosynthesis from a red leaf mutant of Populus deltoids, which accumulated more anthocyanins in the leaves and showed higher expression levels of anthocyanin biosynthesis genes than did the wild type. Gene expression analyses of all TFs regulating anthocyanin biosynthesis demonstrated that only a MYB118 homologous gene, PdMYB118, was up-regulated in the mutant compared with the wide type. Subcellular localization analyses in poplar leaf mesophyll protoplasts showed that PdMYB118-YFP fusion protein was specifically located in nucleus. When transiently expressed in poplar leaf protoplasts, PdMYB118 specifically promoted the expression of anthocyanidin biosynthesis genes. Dual-luciferase assays revealed that PdMYB118 can directly activate the promoters of these genes. When overexpressed in Shanxin Yang (P. davidiana × P. bolleana), a hybrid clone commercially grown for landscaping in the northern part of China, transgenic plants overexpressing PdMYB118 produced more anthocyanins in the leaves and turned their color into redness when grown in both greenhouse and field. Consistently, transcripts of some important anthocyanidin biosynthesis genes were significantly increased in the leaves of transgenic plants. All these results indicate that PdMYB118 functions as an essential transcription factor regulating anthocyanin biosynthesis in poplar and could be used for the genetic engineering of colorful tree species.

Maize VKS1 Regulates Mitosis and Cytokinesis During Early Endosperm Development.

Cell number is a critical factor that determines kernel size in maize (Zea mays). Rapid mitotic divisions in early endosperm development produce most of the cells that make up the starchy endosperm; however, the mechanisms underlying early endosperm development remain largely unknown. We isolated a maize mutant that shows a varied-kernel-size phenotype (vks1). Vks1 encodes ZmKIN11, which belongs to the kinesin-14 subfamily and is predominantly expressed in early endosperm development. VKS1 dynamically localizes to the nucleus and microtubules and plays key roles in the migration of free nuclei in the coenocyte as well as in mitosis and cytokinesis in early mitotic divisions. Absence of VKS1 has relatively minor effects on plants but causes deformities in spindle assembly, sister chromatid separation, and phragmoplast formation in early endosperm development, thereby resulting in reduced cell proliferation. Severities of aberrant mitosis and cytokinesis within individual vks1 endosperms differ, thereby resulting in varied kernel sizes. Our discovery highlights VKS1 as a central regulator of mitosis in early maize endosperm development and provides a potential approach for future yield improvement.

Fasciclin-like arabinogalactan proteins, PtFLAs, play important roles in GA-mediated tension wood formation in Populus.

In Populus, the transcripts of fasciclin-like arabinogalactan proteins (FLAs) are accumulated in tension wood (TW) xylem, however their biological functions in TW formation are largely unknown. In this work, we demonstrated that PtFLA6, one of poplar TW-associated PtFLAs, was abundantly expressed in TW, and mainly localized in differentiating G-fibers. The bended stems of PtFLA6 antisense transgenic poplar showed decreased transcripts of PtFLAs, including PtFLA6, and reduced PtFLA6 like proteins, leading to inhibited TW differentiation and formation. We also showed that gibberellin A3 (GA3) was enriched in the xylem of TW side, accompanied with a lowered level of PtRGA1, a poplar DELLA protein. When GA3 biosynthesis was restrained in the bended poplar stems by a GA biosynthesis inhibitor (daminozide), TW formation was obviously repressed, as a result of restricted PtRGA1 degradation, and reduced PtFLA6 like proteins and PtFLA expression. Further studies indicated that PtFLAs were negatively regulated by PtRGA1. This study suggests that PtFLAs play important roles in the poplar TW formation, possibly regulated by GA signaling.

Overexpression of Populus trichocarpa CYP85A3 promotes growth and biomass production in transgenic trees.

Brassinosteroids (BRs) are essential hormones that play crucial roles in plant growth, reproduction and response to abiotic and biotic stress. In Arabidopsis, AtCYP85A2 works as a bifunctional cytochrome P450 monooxygenase to catalyse the conversion of castasterone to brassinolide, a final rate-limiting step in the BR-biosynthetic pathway. Here, we report the functional characterizations of PtCYP85A3, one of the three AtCYP85A2 homologous genes from Populus trichocarpa. PtCYP85A3 shares the highest similarity with AtCYP85A2 and can rescue the retarded-growth phenotype of the Arabidopsis cyp85a2-2 and tomato dx mutants. Constitutive expression of PtCYP85A3, driven by the cauliflower mosaic virus 35S promoter, increased the endogenous BR levels and significantly promoted the growth and biomass production in both transgenic tomato and poplar. Compared to the wild type, plant height, shoot fresh weight and fruit yield increased 50%, 56% and 43%, respectively, in transgenic tomato plants. Similarly, plant height and stem diameter increased 15% and 25%, respectively, in transgenic poplar plants. Further study revealed that overexpression of PtCYP85A3 enhanced xylem formation without affecting the composition of cellulose and lignin, as well as the cell wall thickness in transgenic poplar. Our finding suggests that PtCYP85A3 could be used as a potential candidate gene for engineering fast-growing trees with improved wood production.

Ectopic expression of a cytochrome P450 monooxygenase gene PtCYP714A3 from Populus trichocarpa reduces shoot growth and improves tolerance to salt stress in transgenic rice.

In Arabidopsis thaliana and Oryza sativa, the cytochrome P450 (CYP) 714 protein family represents a unique group of CYP monooxygenase, which functions as a shoot-specific regulator in plant development through gibberellin deactivation. Here, we report the functional characterizations of PtCYP714A3, an OsCYP714D1/Eui homologue from Populus trichocarpa. PtCYP714A3 was ubiquitously expressed with the highest transcript level in cambium-phloem tissues, and was greatly induced by salt and osmotic stress in poplar. Subcellular localization analyses indicated that PtCYP714A3-YFP fusion protein was targeted to endoplasmic reticulum (ER). Expression of PtCYP714A3 in the rice eui mutant could rescue its excessive-shoot-growth phenotype. Ectopic expression of PtCYP714A3 in rice led to semi-dwarfed phenotype with promoted tillering and reduced seed size. Transgenic lines which showed significant expression of PtCYP714A3 also accumulated lower GA level than did the wild-type (WT) plants. The expression of some GA biosynthesis genes was significantly suppressed in these transgenic plants. Furthermore, transgenic rice plants exhibited enhanced tolerance to salt and maintained more Na(+) in both shoot and root tissues under salinity stress. All these results not only suggest a crucial role of PtCYP714A3 in shoot responses to salt toxicity in rice, but also provide a molecular basis for genetic engineering of salt-tolerant crops.

GhCFE1A, a dynamic linker between the ER network and actin cytoskeleton, plays an important role in cotton fibre cell initiation and elongation.

Fibre cell initiation and elongation is critical for cotton fibre development. However, little is known about the regulation of initiation and elongation during fibre cell development. Here, the regulatory role of a novel protein GhCFE1A was uncovered. GhCFE1A is preferentially expressed at initiation and rapid elongation stages during fibre development; in addition, much higher expression of GhCFE1A was detected at the fibre initiation stage in fibreless cotton mutants than in the fibre-bearing TM-1 wild-type. Importantly, overexpression of GhCFE1A in cotton not only delayed fibre cell elongation but also significantly reduced the density of lint and fuzz fibre initials and stem trichomes. Yeast two-hybrid assay showed that GhCFE1A interacted with several actin proteins, and the interaction was further confirmed by co-sedimentation assay. Interestingly, a subcellular localization assay showed that GhCFE1A resided on the cortical endoplasmic reticulum (ER) network and co-localized with actin cables. Moreover, the density of F-actin filaments was shown to be reduced in GhCFE1A-overexpressing fibres at the rapid elongation stage compared with the wild-type control. Taken together, the results demonstrate that GhCFE1A probably functions as a dynamic linker between the actin cytoskeleton and the ER network, and plays an important role in fibre cell initiation and elongation during cotton fibre development.

Antisense expression of the fasciclin-like arabinogalactan protein FLA6 gene in Populus inhibits expression of its homologous genes and alters stem biomechanics and cell wall composition in transgenic trees.

Fasciclin-like arabinogalactan proteins (FLAs) play important roles in the growth and development of roots, stems, and seeds in Arabidopsis. However, their biological functions in woody plants are largely unknown. In this work, we investigated the possible function of PtFLA6 in poplar. Quantitative real-time PCR, PtFLA6-yellow fluorescent protein (YFP) fusion protein subcellular localization, Western blotting, and immunohistochemical analyses demonstrated that the PtFLA6 gene was expressed specifically in the xylem of mature stem, and PtFLA6 protein was distributed ubiquitous in plant cells and accumulated predominantly in stem xylem fibres. Antisense expression of PtFLA6 in the aspen hybrid clone Poplar davidiana×Poplar bolleana reduced the transcripts of PtFLA6 and its homologous genes. Transgenic plants that showed a significant reduction in the transcripts of PtFLAs accumulated fewer PtFLA6 and arabinogalactan proteins than did the non-transgenic plants, leading to reduced stem flexural strength and stiffness. Further studies revealed that the altered stem biomechanics of transgenic plants could be attributed to the decreased cellulose and lignin composition in the xylem. In addition expression of some xylem-specific genes involved in cell wall biosynthesis was downregulated in these transgenic plants. All these results suggest that engineering the expression of PtFLA6 and its homologues could modulate stem mechanical properties by affecting cell wall composition in trees.

Functional repression of PtSND2 represses growth and development by disturbing auxin biosynthesis, transport and signaling in transgenic poplar.

Using chimeric repressor silencing technology, we previously reported that functional repression of PtSND2 severely arrested wood formation in transgenic poplar (Populus). Here, we provide further evidence that auxin biosynthesis, transport and signaling were disturbed in these transgenic plants, leading to pleiotropic defects in their growth patterns, including inhibited leaf enlargement and vascular tissue development in the leaf central vein, suppressed cambial growth and fiber elongation in the stem, and arrested growth in the root system. Two transgenic lines, which displayed the most remarkable phenotypic deviation from the wild-type, were selected for detailed studies. In both transgenic lines, expression of genes for auxin biosynthesis, transport and signaling was down-regulated, and indole-3-acetic acid distribution was severely disturbed in the apical buds, leaves, stems and roots of field-grown transgenic plants. Transient transcription dual-luciferase assays of ProPtTYDC2::LUC, ProPttLAX2::LUC and ProPoptrIAA20.2::LUC in poplar protoplasts revealed that expression of auxin-related genes might be regulated by PtSND2 at the transcriptional level. All these results indicate that functional repression of PtSND2 altered auxin biosynthesis, transport and signaling, and thereby disturbed the normal growth and development of transgenic plants.

Discovery and identification of a novel Ligon lintless-like mutant (Lix) similar to the Ligon lintless (Li1) in allotetraploid cotton.

Mutants are a powerful resource for studying gene structure, function, and evolution. In this present study, a novel Ligon lintless-like mutant (Lix), that has short fibers and deformed leaves and stems, was isolated from the progeny of transgenic cottons. The Lix mutant is similar in morphology to the Ligon lintless (Li1) mutant. Genetic analysis and molecular mapping were performed for the Lix and Li1 mutants. These two mutants are monogenic dominant mutants with distorted growth of vegetative and reproductive structures. Seedlings of the dominant homozygote Li 1 Li 1 genotype are lethal, while LixLix plants are viable but show no reproductive growth. Molecular tagging showed that the Lix gene is located on Chr. 04 in a 30.9-cM region spanned by NAU8376 and NAU3469. In a previous study, the Li 1 gene was mapped to Chr. 22, and Chr. 04 and Chr. 22 are homoelogous chromosomes in tetraploid cotton. So, we propose that Lix and Li1 mutants have similar mutated morphology, and Lix is mapped to a homoelogous chromosome carrying Li 1 . The identification and genetic mapping of Lix/Li 1 genes using mutants provides a foundation for isolating these genes. In turn, this will permit studies to elucidate the functional and evolutionary roles for these genes in cotton growth and development.