sparse inflorescence1, barren inflorescence1 and barren stalk1 Promote Cell Elongation in Maize Inflorescence Development

Abstract

The sparse inflorescence1 (spi1), Barren inflorescence1 (Bif1), barren inflorescence2 (bif2), and barren stalk1 (ba1) mutants produce fewer branches and spikelets in the inflorescence due to defects in auxin biosynthesis, transport, or response. We report that spi1, bif1, and ba1, but not bif2, also function in promoting cell elongation in the inflorescence.

AUXIN is essential for lateral organ and axillary meristem initiation in plants (Barazesh and McSteen 2008b; Delker et al. 2008). The maize (Zea mays) mutants, sparse inflorescence1 (spi1), Barren inflorescence1 (Bif1), barren inflorescence2 (bif2), and barren stalk1 (ba1) produce fewer branches and spikelets in the inflorescence due to defects in axillary meristem initiation (McSteen and Hake 2001; Ritter et al. 2002; Barazesh and McSteen 2008a; Gallavotti et al. 2008). spi1 functions in localized auxin biosynthesis, while bif1 and bif2 regulate auxin transport (McSteen et al. 2007; Barazesh and McSteen 2008a; Gallavotti et al. 2008). spi1; bif2 and Bif1; bif2 double mutants have a synergistic interaction producing dwarf plants with fewer leaves, indicating that spi1, bif1, and bif2 also function in leaf initiation during vegetative development (Barazesh and McSteen 2008a; Gallavotti et al. 2008). Synergistic interactions between mutants affecting auxin biosynthesis and auxin transport have also been reported in Arabidopsis (Arabidopsis thaliana) (Cheng et al. 2007a,b).

Investigation of tassel-length reduction in spi1 mutants:

An interesting aspect of the spi1 phenotype is that the length of the tassel (male inflorescence) is reduced compared to a normal tassel (Figure 1, A and F). Previous analysis revealed that spikelets grow over the tip of the tassel (arrowhead in Figure 1C) (Gallavotti et al. 2008). Development of spikelets over the tip of the tassel could consume the apical inflorescence meristem, which would inhibit growth of the tassel. To test whether the production of spikelets over the tip causes the short inflorescence phenotype, we utilized spi1; bif2 double mutants, which produce tassels with no spikelets (Figure 1A) (Gallavotti et al. 2008). SEM analysis verified that spi1; bif2 mutants fail to initiate spikelet pair meristems (SPMs) (Figure 1, B–E). However, there was no significant difference in the tassel length of spi1; bif2 double mutants compared to spi1 single mutants (Figure 1F, P = 0.366), showing that the growth of spikelets over the tip of the inflorescence does not the cause the reduction in tassel length in spi1 mutants.

Figure 1.—

Genetic interaction of spi1 with bif2. Double mutants were constructed in the B73 background with bif2-77 and spi1-ref alleles, which were genotyped as previously described (Gallavotti et al. 2008). For analysis of immature spi1; bif2 double mutants, tassels were dissected from 5-week-old plants, and fixation and SEM was carried out as previously described (Barazesh and McSteen 2008a). For mature plant analysis, all plants were grown in the field to maturity. Two families of 120 kernels were planted at two different field locations. (A) Mature tassel phenotype of all genetic classes in a family segregating for both spi1 and bif2. (B–E) SEM analysis of developing inflorescences of (B) normal, (C) spi1, (D) bif2, and (E) spi1;bif2 double mutants. Arrowhead indicates spikelets growing over the tip of the spi1 mutant tassel. IM, inflorescence meristem; SPM, spikelet pair meristem. Bar, 100 μm. (F) Mature tassel length of all genetic classes in a family segregating for both spi1 and bif2. Tassel length was measured from the node at the base of the flag leaf to the tip of the tassel. Sample size was 10 for each genetic class.

spi1, bif1, and ba1 function in cell elongation in the tassel:

To determine if the reduced tassel length in spi1 mutants was due to defective cell elongation, impressions were taken of epidermal cells of mature spi1 tassels, and cell length was quantified. Cell length was significantly decreased in the epidermal cells of spi1 tassels compared to normal (Figure 2, Table 1). However, cell length in the epidermis of the leaf was unaffected (data not shown).

Figure 2.—

spi1, Bif1, and ba1 have reduced cell elongation. Nail polish impressions of epidermal cells from the base of the mature tassel in (A) normal, (B) spi1, (C) Bif1, and (D) ba1. Bar, 100 μm.

TABLE 1.

spi1, Bif1, and ba1 affect cell elongation

Genotype	Mean cell length (μm)a	±SE	N	P-valueb
Normal	156.51	6.01	5	—
spi1	127.63	6.81	5	0.015
bif2	157.83	8.36	5	0.902
Bif1	109.66	7.83	7	0.001
ba1	101.86	4.80	7	<0.001

To measure the length of epidermal cells of mature tassels, clear nail polish was used to make impressions from the surface of the tassel rachis. Double-sided tape was used to lift the nail polish from the surface of the tassel and adhere it to a slide. Impressions were viewed at ×20 magnification using a Nikon 80i microscope and photographed with a Nikon DM1200F camera. Approximately 25 cells/biological replicate were measured in three regions of the tassel. Similar results were obtained for each region, and data for one region near the base are shown. SE, standard error of mean; N, number of biological replicates.

^aMean calculated for each biological replicate and then for each genotype.

^bP-value indicates the significance of the difference between the mutant and the normal calculated using a Student's t test.

The reduced tassel length of spi1 prompted us to investigate if other barren inflorescence mutants had this defect. We discovered that bif2 did not affect tassel length (Figure 1F) or cell elongation (Table 1). However, both Bif1 and ba1 mutants had shorter tassels than normal (Figure 3, Table 2, and Table 3), and epidermal cell length was significantly reduced (Figure 2, Table 1). As Bif1 and ba1 affected tassel length, we investigated the interaction between spi1 and each of these mutants.

Figure 3.—

Genetic interaction of spi1 with Bif1 and of spi1 with ba1. (A) Mature tassel phenotype of a spi1, Bif1 segregating family. Double mutants were constructed in the B73 genetic background with the spi1-ref and Bif1-N1440 alleles (Barazesh and McSteen 2008a; Gallavotti et al. 2008). Plants were genotyped for the spi1-ref allele as reported (Gallavotti et al. 2008). A total of 120 plants were analyzed in two different field locations. (B) Mature tassel phenotype of a spi1, ba1 segregating family. Double mutants were constructed in the B73 genetic background with the spi1-ref and ba1-ref alleles and genotyped as described (Barazesh and McSteen 2008a; Gallavotti et al. 2008). A total of 120 plants were analyzed in two different field locations. (C) Close-up of the surface of the tassel rachis showing prominent bract leaf primordia in ba1 (arrowhead), which are not present in spi1; ba1.

TABLE 2.

spi1; Bif1 double-mutant analysis

Genotype	Tassel length (cm)	Branch no.	Spikelet no.	Plant height (cm)	Leaf no.
Normal	52.73 ± 1.74	8.46 ± 0.57	685.1 ± 23	215.91 ± 6.48	22.90 ± 0.21
Bif1/+	48.5 ± 0.9a	2.67 ± 0.31a	112.17 ± 8.32a	179.24 ± 6.02a	21.53 ± 0.34a
Bif1/Bif1	44.33 ± 1.55a	0.556 ± 0.18a	5.667 ± 0.83a	161.56 ± 6.59a	21 ± 0.37a
spi1	31.79 ± 0.86a	4.43 ± 0.42a	68.71 ± 7.86a	152.13 ± 9.43a	21.62 ± 0.50a
spi1; Bif1/+	31.5 ± 0.97b	2.286 ± 0.27c	29.5 ± 4.15bc	148.11 ± 5.3	20.67 ± 0.40
spi1; Bif1/Bif1	30.89 ± 0.84b	0.11 ± 0.11c	0.22 ± 0.22bc	143.64 ± 3.99b	20.18 ± 0.53

For quantification of inflorescence characters, the sample size was normal 13, Bif1/+ 12, Bif1/Bif1 9, spi1 14, Bif1/+; spi1/spi1 14, and Bif1/Bif1; spi1/spi1 9. For quantification of vegetative characters, the sample size was Normal 11, Bif1/+ 17, Bif1/Bif1 9, spi1 4, Bif1/+; spi1/spi1 6, and Bif1/Bif1; spi1/spi1 6.

^aValue is significantly different from normal, P < 0.05.

^bValue is significantly different from Bif1/+ and Bif1/Bif1, P < 0.05.

^cValue is significantly different from spi1, P < 0.05.

TABLE 3.

spi1; ba1 double-mutant analysis

Genotype	Tassel length (cm)	Branch no.	Spikelet no.	Ear no.
Normal	52.32 ± 1.01	8 ± 0.30	646.7 ± 12.6	1.71 ± 0.07
spi1	32.32 ± 1.2a	3.18 ± 0.32a	36.36 ± 3.59a	1.38 ± 0.15a
ba1	36.8 ± 1.18a	0 ± 0a	21.7 ± 3.24a	0 ± 0a
spi1; ba1	25.1 ± 1.8b	0 ± 0	1.5 ± 1.19b	0 ± 0

For quantification of inflorescence characters, the sample size was normal 11, spi1 12, ba1 11, and spi1;ba1 10. For quantification of ear number, the sample size was normal 56, spi1 16, ba1 24, and spi1;ba1 7.

^aValue is significantly different from normal, P < 0.05.

^bValue is significantly different from either single mutant, P < 0.05.

spi1 interaction with Bif1:

spi1; Bif1 double mutants had a severe tassel phenotype, with no tassel branches and very few spikelets, similar to the spi1; bif2 inflorescence phenotype (Figure 3A, Table 2) (Gallavotti et al. 2008). However, the tassel length defect in spi1; Bif1 was not statistically different from spi1 single mutants (P = 0.464), suggesting that spi1 and Bif1 may function in the same pathway to promote tassel length. Unlike the spi1; bif2 double mutants (Gallavotti et al. 2008), the spi1; Bif1 double mutants did not have a synergistic effect on vegetative development (Table 2). Plant height and leaf number were not significantly different in spi1; Bif1 double mutants compared to spi1 single mutants (P = 0.429 and 0.066, respectively).

spi1 interaction with ba1:

The spi1; ba1 double mutant was similar to ba1 single mutants, with no ears and no tassel branches (Figure 3B, Table 3). The reduction in spikelet number in the tassel was more severe than either spi1 (P < 0.0001) or ba1 single mutants (P < 0.001). Furthermore, the double-mutant tassels were more severely reduced in length than either spi1 (P < 0.005) or ba1 single mutants (P < 0.001). We infer that spi1 and ba1 play independent roles in spikelet formation and tassel elongation although, as neither of these mutants are known to be null alleles, it is also possible that they function in the same pathway.

ba1 mutants produce a regular pattern of bumps on the surface of the tassel rachis, which are the bract leaf primordia that subtend axillary meristems in the tassel (Figure 3C) (Ritter et al. 2002). The surface of the spi1; ba1 tassel rachis was smooth, similar to that of the spi1 single mutant, indicating that the bract leaf bumps were missing (Figure 3C). Similarly, the Bif1; ba1 and bif2; ba1 double mutants had a smooth tassel rachis (Barazesh and McSteen 2008a; Skirpan et al. 2008). Therefore, both auxin biosynthesis and transport are required for bract leaf initiation.

Conclusions:

Auxin is known to function in cell expansion (Jones et al. 1998; Christian et al. 2006). A link between auxin biosynthesis and cell expansion was illustrated by experiments involving the erecta (er) mutants of Arabidopsis, which are defective in internode and pedicel elongation (Woodward et al. 2005). Overexpression of the auxin biosynthesis gene, YUC5, suppressed the er phenotype by increasing the elongation of epidermal pavement cells, showing that an increase in localized auxin biosynthesis led to an increase in cell elongation. In this article, we have shown that a decrease in localized auxin biosynthesis led to a decrease in cell elongation, with spi1 epidermal cells significantly reduced in length compared to normal. Mutations in other auxin biosynthesis genes in Arabidopsis and petunia (Petunia inflata) also cause short inflorescences (Tobena-Santamaria et al. 2002; Cheng et al. 2006; Stepanova et al. 2008), implying that these mutations may also affect cell elongation.

spi1 is expressed in a very restricted pattern in the inflorescence (Gallavotti et al. 2008). As spi1 appears to function in tissues in which the gene is not expressed, we infer that auxin synthesized by spi1 is transported rapidly to other cells and therefore that spi1 functions in a non-cell-autonomous manner. This is consistent with the finding that a homologous gene in Petunia acts non-cell autonomously (Tobena-Santamaria et al. 2002).

Previously, it was shown that auxin transport functions in cell elongation during vegetative development (Multani et al. 2003). Here, we show that spi1, Bif1, and ba1 mutants also have defects in cell elongation in the inflorescence. This emphasizes the importance of both auxin biosynthesis and transport in cell elongation during inflorescence development.