Qin-Bao Li

Structural organization of the spinach endoplasmic reticulum-luminal 70-kilodalton heat-shock cognate gene and expression of 70-kilodalton heat-shock genes during cold acclimation.

The 70-kD heat-shock proteins (HSP70s) are encoded by a multigene family in eukaryotes. In plants, the 70-kD heat-shock cognate (HSC70) proteins are located in organellar and cytosolic compartments of cells in most tissues. Previous work has indicated that HSC70 proteins of spinach (Spinacia oleracea) are actively synthesized during cold-acclimating conditions. We have isolated, sequenced, and characterized cDNA and genomic clones for the endoplasmic reticulum (ER) luminal HSC70 protein (immunoglobulin heavy chain-binding protein; BiP) of spinach. The spinach ER-luminal HSC70 is a constitutively expressed gene consisting of eight exons. Spinach BiP mRNA appears to be up-regulated during cold acclimation but is not expressed during water stress or heat shock. In contrast to the differential regulation of mRNA, the ER-luminal HSC70 protein levels remain constant in response to various environmental stresses. Two other members of the spinach 70-kD heat-shock (HS70) multigene family also show differential expression in response to a variety of environmental stresses. A constitutively expressed cytosolic HSC70 protein in spinach appears also to be up-regulated in response to both cold-acclimating and heat-shock treatments. Spinach also contains a cold-shock-induced HS70 gene that is not expressed during heat shock or water stress. Since HSP70s are considered to be involved with the chaperoning and folding of proteins, the data further support the concept that they may be important for maintaining cellular homeostasis and proper protein biogenesis during cold acclimation of spinach.

Phylogeny of the American Amaryllidaceae based on nrDNA ITS sequences

Abstract Analysis of three plastid DNA sequences for a broad sampling of Amaryllidaceae resolve the American genera of the Amaryllidaceae as a clade that is sister to the Eurasian genera of the family, but base substitution rates for these genes are too low to resolve much of the intergeneric relationships within the American clade. We obtained ITS rDNA sequences for 76 species of American Amaryllidaceae and analyzed the aligned matrix cladistically, both with and without gaps included, using two species of Pancratium as outgroup taxa. ITS resolves two moderately to strongly supported groups, an Andean tetraploid clade, and a primarily extra-Andean “hippeastroid” clade. Within the hippeastroid clade, the tribe Griffineae is resolved as sister to the rest of Hippeastreae. The genera Rhodophiala and Zephyranthes are resolved as polyphyletic, but the possibility of reticulation within this clade argues against any re-arrangement of these genera without further investigation. Within the Andean subclade, Eustephieae resolves as sister to all other tribes; a distinct petiolate-leafed group is resolved, combining the tribe Eucharideae and the petiolate Stenomesseae; and a distinct Hymenocallideae is supported. These Andean clades are all at least partially supported by plastid sequence data as well. We infer from our data that a great deal of the diversity of the family in the Americas is recent, and that the American Amaryllidaceae may have been reduced to peripheral isolates some time after its initial entry and spread through the Americas. While the sister relationship of the American and Eurasian clades might argue for a Boreotropical origin for the family in America, the cladistic relationships within the American clade based on ITS do not provide any further support for this or any other hypothesis of the familys entry into America. The new tribe Clinantheae is described (four genera: Clinanthus, Pamianthe, Paramongaia, and Pucara), and the lorate-leafed species of Stenomesson are transferred to Clinanthus. Communicating Editor: Kathleen A. Kron

Viviparous1 Alters Global Gene Expression Patterns through Regulation of Abscisic Acid Signaling

Maize (Zea mays) Viviparous1 (VP1) and Arabidopsis ABI3 are orthologous transcription factors that regulate key aspects of plant seed development and ABA signaling. To understand VP1-regulated gene expression on a global scale, we have performed oligomicroarray analysis of transgenic Arabidopsis carrying 35S::VP1 in an abi3 null mutant background. We have identified 353 VP1/ABA-regulated genes by GeneChip analysis. Seventy-three percent of the genes were affected by both VP1 and ABA in vegetative tissues, indicating a tight coupling between ABA signaling and VP1 function. A large number of seed-specific genes were ectopically expressed in vegetative tissue of 35S::VP1 plants consistent with evidence that VP1 and ABI3 are key determinants of seed-specific expression. ABI5, a positive regulator of ABA signaling, was activated by VP1, indicating conservation of the feed-forward pathway mediated by ABI3. ABA induction of ABI1 and ABI2, negative regulators of ABA signaling, was strongly inhibited by VP1, revealing a second pathway of feed-forward regulation. These results indicate that VP1 strongly modifies ABA signaling through feed-forward regulation of ABI1/ABI5-related genes. Of the 32 bZIP transcription factors represented on the GeneChip, genes in the ABI5 clade were specifically coregulated by ABA and VP1. Statistical analysis of 5′ upstream sequences of the VP1/ABA-regulated genes identified consensus abscisic responsive elements as an enriched element, indicating that many of the genes could be direct targets of the ABI5-related bZIPs. The Sph element is an enriched sequence motif in promoters of genes co-activated by ABA and VP1 but not in promoters of genes activated by ABA alone. This analysis reveals that distinct combinatorial patterns of promoter elements distinguish subclasses of VP1/ABA coregulated genes.

The Organization and Evolution of the Spinach Stress 70 Molecular Chaperone Gene Family

The stress 70 molecular chaperones of plants are localized and function in all of the major subcellular compartments of the cell. Collectively, all of the various forms are encoded by a multigene family in the nucleus. At least 12 members of this family have been found, and sequence and DNA blot analyses provide an emerging description of the diversity of gene structure organization for this family of evolutionarily conserved proteins in spinach. They exhibit not only structural diversity in the organization of coding and noncoding regions but also distinct expression patterns for different tissues and abiotic conditions. The results of phylogenetic analyses are concordant with at least four major evolutionary events that gave rise to stress 70 molecular chaperones in each of four major subcellular compartments of plant cells: the plastid, mitochondrion, cytoplasm, and endoplasmic reticulum. The varied expression patterns also illustrate the complexity of effectively interpreting the role of any one of these stress-related proteins in response to abiotic stress in the absence of context to the other members of the family.

Systematics of Amaryllidaceae based on cladistic analysis of plastid sequence data

Cladistic analyses of plastid DNA sequences rbcL and trnL-F are presented separately and combined for 48 genera of Amaryllidaceae and 29 genera of related asparagalean families. The combined analysis is the most highly resolved of the three and provides good support for the monophyly of Amaryllidaceae and indicates Agapanthaceae as its sister family. Alliaceae are in turn sister to the Amaryllidaceae/Agapanthaceae clade. The origins of the family appear to be western Gondwanaland (Africa), and infrafamilial relationships are resolved along biogeographic lines. Tribe Amaryllideae, primarily South African, is sister to the rest of Amaryllidaceae; this tribe is supported by numerous morphological synapomorphies as well. The remaining two African tribes of the family, Haemantheae and Cyrtantheae, are well supported, but their position relative to the Australasian Calostemmateae and a large clade comprising the Eurasian and American genera, is not yet clear. The Eurasian and American elements of the family are each monophyletic sister clades. Internal resolution of the Eurasian clade only partially supports currently accepted tribal concepts, and few conclusions can be drawn on the relationships of the genera based on these data. A monophyletic Lycorideae (Central and East Asian) is weakly supported. Galanthus and Leucojum (Galantheae pro parte) are supported as sister genera by the bootstrap. The American clade shows a higher degree of internal resolution. Hippeastreae (minus Griffinia and Worsleya) are well supported, and Zephyranthinae are resolved as a distinct subtribe. An Andean clade marked by a chromosome number of 2n = 46 (and derivatives thereof) is resolved with weak support. The plastid DNA phylogenies are discussed in the context of biogeography and character evolution in the family.

Genetic control of cell wall invertases in developing endosperm of maize

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

Coordinate and non-coordinate expression of the stress 70 family and other molecular chaperones at high and low temperature in spinach and tomato

Stress 70 molecular chaperones are found in all the major subcellular compartments of plant cells, and they are encoded by a multigene family. Twelve members of this family have been identified in spinach. The expression of the stress 70 molecular chaperones in response to heat shock is well-known and it appears that low temperature exposure can also stimulate their expression. However, it has been difficult to determine which member(s) of the family are specifically responsive to low temperature. This study was initiated to determine the levels of expression of the stress 70 family members and other selected chaperones in response to high and low temperature exposure. During heat shock of spinach, of the 10 stress 70 family members that were examined, all 10 showed increased RNA levels after one hour, and all showed down-regulation at longer durations of high temperature exposure. However, the response to low temperature was quite variable and complex. Some members were induced, some were transiently up-regulated, while others showed sustained up-regulation at a low non-freezing temperature. In comparison, the entirety of the molecular chaperone expression response of cold-sensitive tomato at the same low non-freezing temperature was even more dramatic with 11 of 15 molecular chaperones tested exhibiting elevated expression. The increased chaperone expression is consistent with the hypothesis that the biogenesis or stability of some proteins is compromised at low non-freezing temperatures. In contrast, mild freezing sufficient to cause injury of spinach did not materially activate chaperone expression.

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

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

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

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

Sugar–Hormone Cross-Talk in Seed Development: Two Redundant Pathways of IAA Biosynthesis Are Regulated Differentially in the Invertase-Deficient miniature1 (mn1) Seed Mutant in Maize

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